Interfering RNA molecules

ABSTRACT

The present invention is related to a ribonucleic acid comprising a double stranded structure whereby the double-stranded structure comprises a first strand and a second strand, whereby the first strand comprises a first stretch of contiguous nucleotides and whereby said first stretch is at least partially complementary to a target nucleic acid, and the second strand comprises a second stretch of contiguous nucleotides whereby said second stretch is at least partially identical to a target nucleic acid, and whereby the double stranded structure is blunt ended.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/977,710, filed Dec. 22, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/578,636, filed on Dec. 22, 2014, now U.S. Pat.No. 9,222,092, issued Dec. 29, 2015, which is a continuation of U.S.patent application Ser. No. 13/692,178, filed on Dec. 3, 2012, now U.S.Pat. No. 8,933,215, issued Jan. 13, 2015, which is a continuation ofU.S. patent application Ser. No. 12/986,389, filed on Jan. 7, 2011, nowU.S. Pat. No. 8,324,370, issued Dec. 4, 2012, which is a continuation ofU.S. patent application Ser. No. 12/200,296, filed on Aug. 28, 2008, nowU.S. Pat. No. 7,893,245, issued Feb. 22, 2011, which is a continuationof U.S. patent application Ser. No. 10/633,630, filed on Aug. 5, 2003,now U.S. Pat. No. 7,452,987, issued Nov. 18, 2008, which claims thebenefit of U.S. Provisional Application No. 60/402,541, filed Aug. 12,2002, which claims priority to European application No. 02017601.2,filed Aug. 5, 2002, and European application No. 03008383.6, filed Apr.10, 2003. Each of these applications are incorporated herein byreference in their entirety, including all figures, tables and aminoacid or nucleic acid sequences.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 680132000106seqlist.txt,date recorded: May 1, 2017, size: 85 KB).

FIELD OF THE INVENTION

The invention provides novel forms of interfering ribonucleic acidmolecules having a double-stranded structure. The first strand comprisesa first stretch of contiguous nucleotides that is at least partiallycomplementary to a target nucleic acid, and the second strand comprisesa second stretch of contiguous nucleotides that is at least partiallyidentical to a target nucleic acid. Methods for using these molecules,for example for inhibiting expression of a target gene, andpharmaceutical compositions, cells and organisms containing thesemolecules also are provided.

BACKGROUND OF THE INVENTION

RNA-mediated interference (RNAi) is a post-transcriptional genesilencing mechanism initiated by double stranded RNA (dsRNA) homologousin sequence to the silenced gene (Fire (1999), Trends Genet 15, 358-63,Tuschl, et al. (1999), Genes Dev 13, 3191-7, Waterhouse, et al. (2001),Nature 411, 834-42, Elbashir. et al. (2001), Nature 411, 494-8, forreview see Sharp (2001), Genes Dev 15, 485-90, Barstead (2001), CurrOpin Chem Biol 5, 63-6). RNAi has been used extensively to determinegene function in a number of organisms, including plants (Baulcombe(1999), Curr Opin Plant Biol 2, 109-13), nematodes (Montgomery, et al.(1998), Proc Natl Acad Sci USA 95, 15502-7), Drosophila (Kennerdell. etal. (1998), Cell 95, 1017-26, Kennerdell, et al. (2000), Nat Biotechnol18, 896-8). In the nematode C. elegans about one third of the genome hasalready been subjected to functional analysis by RNAi (Kim (2001), CurrBiol 11, R85-7, Maeda. et al. (2001), Curr Biol 11, 171-6).

Until recently RNAi in mammalian cells was not generally applicable,with the exception of early mouse development (Wianny. et al. (2000),Nat Cell Biol 2, 70-5). The discovery that transfection of duplexes of21-nt into mammalian cells interfered with gene expression and did notinduce a sequence independent interferon-driven anti-viral responseusually obtained with long dsRNA led to new potential application indifferentiated mammalian cells (Elbashir et al. (2001), Nature 411,494-8). Interestingly these small interfering RNAs (siRNAs) resemble theprocessing products from long dsRNAs suggesting a potential bypassingmechanism in differentiated mammalian cells. The Dicer complex, a memberof the RNAse II family, necessary for the initial dsRNA processing hasbeen identified (Bernstein. et al. (2001), Nature 409, 363-6, Billy. etal. (2001), Proc Natl Acad Sci USA 98, 14428-33). One of the problemspreviously encountered when using unmodified ribooligonucleotides wasthe rapid degradation in cells or even in the serum-containing medium(Wickstrom (1986), J Biochem Biophys Methods 13, 97-102, Cazenave. etal. (1987), Nucleic Acids Res 15, 10507-21). It will depend on theparticular gene function and assay systems used whether the respectiveknock down induced by transfected siRNA will be maintained long enoughto achieve a phenotypic change.

It is apparent, therefore, that synthetic interfering RNA molecules thatare both stable and active in a biochemical environment such as a livingcell are greatly to be desired.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to providecompositions and methods using interfering RNA molecules having enhancedstability.

In accomplishing this object, there has been provided, in accordancewith a first aspect of the present invention, a ribonucleic acidcomprising a double stranded structure whereby the double-strandedstructure comprises a first strand and a second strand, whereby thefirst strand comprises a first stretch of contiguous nucleotides andwhereby said first stretch is at least partially complementary to atarget nucleic acid, and the second strand comprises a second stretch ofcontiguous nucleotides whereby said second stretch is at least partiallyidentical to a target nucleic acid, and whereby the double strandedstructure is blunt ended.

In accordance with a second aspect of the present invention there hasbeen provided a ribonucleic acid comprising a double stranded structurewhereby the double-stranded structure comprises a first strand and asecond strand, whereby the first strand comprises a first stretch ofcontiguous nucleotides and whereby said first stretch is at leastpartially complementary to a target nucleic acid, and the second strandcomprises a second stretch of contiguous nucleotides, whereby saidsecond stretch is at least partially identical to a target nucleic acid,whereby the first stretch and/or the second stretch have a length of 18or 19 nucleotides.

In an embodiment of the ribonucleic acid according to the first aspectof the invention the first stretch and/or the second stretch have alength of 18 or 19 nucleotides.

In a further embodiment of the ribonucleic acid according to the firstaspect of the invention the double stranded structure is blunt ended onboth sides of the double strand.

In an alternative embodiment of the ribonucleic acid according to thefirst aspect of the invention the double stranded structure is bluntended on the double stranded structure which is defined by the 5′-end ofthe first strand and the 3′-end of the second strand.

In a further alternative embodiment of the ribonucleic acid according tothe first and the second aspect of the invention the double strandedstructure is blunt ended on the double stranded structure which isdefined by the 3′-end of the first strand and the 5′-end of the secondstrand.

In accordance with a third aspect of the present invention there hasbeen provided a ribonucleic acid comprising a double stranded structurewhereby the double-stranded structure comprises a first strand and asecond strand, whereby the first strand comprises a first stretch ofcontiguous nucleotides and whereby said first stretch is at leastpartially complementary to a target nucleic acid, and the second strandcomprises a second stretch of contiguous nucleotides and whereby saidsecond stretch is at least partially identical to a target nucleic acid,and whereby at least one of the two strands has an overhang of at leastone nucleotide at the 5′-end.

In an embodiment of the ribonucleic acid according to the third aspectof the present invention the overhang consists of at least onenucleotide which is selected from the group comprising ribonucleotidesand deoxyribonucleotides

In a more preferred embodiment of the ribonucleic acid according to thethird aspect of the present invention the nucleotide has a modificationwhereby said modification is preferably selected from the groupcomprising nucleotides being an inverted abasic and nucleotides havingan NH₂-modification at the 2′-position.

In a preferred embodiment of the ribonucleic acid according to the thirdaspect of the present invention at least one of the strands has anoverhang of at least one nucleotide at the 3′-end consisting ofribonucleotide or deoxyribonucleotide.

In another preferred embodiment of the ribonucleic acid according to thethird aspect of the present invention the first stretch and/or thesecond stretch have a length of 18 or 19 nucleotides.

In an embodiment of the ribonucleic acid according to any aspect of thepresent invention the double-stranded structure has a length of 17 to 21nucleotides, preferably 18 to 19 nucleotides.

In an embodiment of the ribonucleic acid according to the third aspectof the present invention the overhang at the 5′-end is on the secondstrand.

In a preferred embodiment of the ribonucleic acid according to the thirdaspect of the present invention the first strand comprises also anoverhang, preferably at the 5′-end.

In an embodiment of the ribonucleic acid according to the third aspectof the present invention the 3′-end of the first strand comprises anoverhang.

In an alternative embodiment of the ribonucleic acid according to thethird aspect of the present invention the overhang at the 5′-end is onthe first strand.

In a preferred embodiment thereof the second strand also comprises anoverhang, preferably at the 5′-end.

In an embodiment of the ribonucleic acid according to the third aspectof the present invention the 3′-end of the first strand comprises anoverhang.

In an embodiment of the ribonucleic acid according to any aspect of thepresent invention at least one nucleotide of the ribonucleic acid has amodification at the 2′-position and the modification is preferablyselected from the group comprising amino, fluoro, methoxy, alkoxy andalkyl.

In accordance with a fourth aspect of the present invention there hasbeen provided a ribonucleic acid comprising a double stranded structure,whereby the double-stranded structure comprises a first strand and asecond strand, whereby the first strand comprises a first stretch ofcontiguous nucleotides and whereby said first stretch is at leastpartially complementary to a target nucleic acid, and the second strandcomprises a second stretch of contiguous nucleotides and whereby saidsecond stretch is at least partially identical to a target nucleic acid,whereby said first strand and/or said second strand comprises aplurality of groups of modified nucleotides having a modification at the2′-position whereby within the strand each group of modified nucleotidesis flanked on one or both sides by a flanking group of nucleotideswhereby the flanking nucleotides forming the flanking group ofnucleotides is either an unmodified nucleotide or a nucleotide having amodification different from the modification of the modifiednucleotides.

In an embodiment of the ribonucleic acid according to the fourth aspectof the present invention the ribonucleic acid is the ribonucleic acidaccording to the first, second or third aspect of the present invention.

In a further embodiment of the ribonucleic acid according to the fourthaspect of the present invention said first strand and/or said secondstrand comprise said plurality of modified nucleotides.

In another embodiment of the ribonucleic acid according to the fourthaspect of the present invention said first strand comprises saidplurality of groups of modified nucleotides.

In yet another embodiment of the ribonucleic acid according to thefourth aspect of the present invention said second strand comprises saidplurality of groups of modified nucleotides.

In a preferred embodiment of the ribonucleic acid according to thefourth aspect of the present invention the group of modified nucleotidesand/or the group of flanking nucleotides comprises a number ofnucleotides whereby the number is selected from the group comprising onenucleotide to 10 nucleotides.

In another embodiment of the ribonucleic acid according to the fourthaspect of the present invention the pattern of modified nucleotides ofsaid first strand is the same as the pattern of modified nucleotides ofsaid second strand.

In a preferred embodiment of the ribonucleic acid according to thefourth aspect of the present invention the pattern of said first strandaligns with the pattern of said second strand.

In an alternative embodiment of the ribonucleic acid according to thefourth aspect of the present invention the pattern of said first strandis shifted by one or more nucleotides relative to the pattern of thesecond strand.

In an embodiment of the ribonucleic acid according to the fourth aspectof the present invention the modification is selected from the groupcomprising amino, fluoro, methoxy, alkoxy and alkyl.

In another embodiment of the ribonucleic acid according to the fourthaspect of the present invention the double stranded structure is bluntended.

In a preferred embodiment of the ribonucleic acid according to thefourth aspect of the present invention the double stranded structure isblunt ended on both sides.

In another embodiment of the ribonucleic acid according to the fourthaspect of the present invention the double stranded structure is bluntended on the double stranded structure's side which is defined by the5′-end of the first strand and the 3′-end of the second strand.

In still another embodiment of the ribonucleic acid according to thefourth aspect of the present invention the double stranded structure isblunt ended on the double stranded structure's side which is defined atthe 3′-end of the first strand and the 5′-end of the second strand.

In another embodiment of the ribonucleic acid according to the fourthaspect of the present invention at least one of the two strands has anoverhang of at least one nucleotide at the 5′-end.

In a preferred embodiment of the ribonucleic acid according to thefourth aspect of the present invention the overhang consists of at leastone deoxyribonucleotides.

In a further embodiment of the ribonucleic acid according to the fourthaspect of the present invention at least one of the strands has anoverhang of at least one nucleotide at the 3′-end.

In an embodiment of the ribonucleic acid according to any of the aspectsof the present invention the length of the double-stranded structure hasa length from about 17 to 21 and more preferably 18 or 19 bases.

In another embodiment of the ribonucleic acid according to any of theaspects of the present invention the length of said first strand and/orthe length of said second strand is independently from each otherselected from the group comprising the ranges of from about 15 to about23 bases, 17 to 21 bases and 18 or 19 bases.

In a preferred embodiment of the ribonucleic acid according to any ofthe aspects of the present invention the complementarity between saidfirst strand and the target nucleic acid is perfect.

In an embodiment of the ribonucleic acid according to any of the aspectsof the present invention the duplex formed between the first strand andthe target nucleic acid comprises at least 15 nucleotides wherein thereis one mismatch or two mismatches between said first strand and thetarget nucleic acid forming said double-stranded structure.

In an embodiment of the ribonucleic acid according to any of the aspectsof the present invention, wherein both the first strand and the secondstrand each comprise at least one group of modified nucleotides and atleast one flanking group of nucleotides, whereby each group of modifiednucleotides comprises at least one nucleotide and whereby each flankinggroup of nucleotides comprising at least one nucleotide; with each groupof modified nucleotides of the first strand being aligned with aflanking group of nucleotides on the second strand, whereby the mostterminal 5′ nucleotide of the first strand is a nucleotide of the groupof modified nucleotides, and the most terminal 3′ nucleotide of thesecond strand is a nucleotide of the flanking group of nucleotides.

In a preferred embodiment of the ribonucleic acid according to thefourth aspect, wherein each group of modified nucleotides consists of asingle nucleotide and/or each flanking group of nucleotides consists ofa single nucleotide.

In a further embodiment of the ribonucleic acid according to the fourthaspect, wherein on the first strand the nucleotide forming the flankinggroup of nucleotides is an unmodified nucleotide which is arranged in a3′ direction relative to the nucleotide forming the group of modifiednucleotides, and wherein on the second strand the nucleotide forming thegroup of modified nucleotides is a modified nucleotide which is arrangedin 5′ direction relative to the nucleotide forming the flanking group ofnucleotides.

In another embodiment of the ribonucleic acid according to the fourthaspect, wherein the first strand comprises eight to twelve, preferablynine to eleven, groups of modified nucleotides, and wherein the secondstrand comprises seven to eleven, preferably eight to ten, groups ofmodified nucleotides.

In a preferred embodiment of the ribonucleic acid according to any ofthe aspects of the present invention the target gene is selected fromthe group comprising structural genes, housekeeping genes, transcriptionfactors, motility factors, cell cycle factors, cell cycle inhibitors,enzymes, growth factors, cytokines and tumor suppressors.

In a further embodiment of the ribonucleic acid according to any of theaspects of the present invention the first strand and the second strandare linked by a loop structure.

In a preferred embodiment of the ribonucleic acid according to any ofthe aspects of the present invention the loop structure is comprised ofa non-nucleic acid polymer.

In a preferred embodiment thereof the non-nucleic acid polymer ispolyethylene glycol.

In an alternative embodiment thereof the loop structure is comprised ofa nucleic acid.

In an embodiment of the ribonucleic acid according to any of the aspectsof the present invention the 5′-terminus of the first strand is linkedto the 3-terminus of the second strand.

In a further embodiment of the ribonucleic acid according to any of theaspects of the present invention the 3′-end of the first strand islinked to the 5′-terminus of the second strand.

In accordance with a fifth aspect of the present invention there havebeen provided methods of using a ribonucleic acid according to any ofthe aspects of the present invention for target validation.

In accordance with a sixth aspect of the present invention there havebeen provided medicaments and pharmaceutical compositions containing aribonucleic acid according to any of the aspects of the presentinvention, and methods of making such medicaments and compositions.

In a preferred embodiment of the use according to the sixth aspect ofthe present invention methods are provided for the treatment of adisease or of a condition which is selected from the group comprisingglioblastoma, prostate cancer, breast cancer, lung cancer, liver cancer,colon cancer, pancreatic cancer and leukaemia, diabetes, obesity,cardiovascular diseases, and metabolic diseases.

In accordance with a seventh aspect of the present invention there hasbeen provided a cell, for example a knockdown cell, containing aribonucleic acid according to any of the aspects of the presentinvention.

In accordance with an eighth aspect of the present invention there hasbeen provided an organism, for example a knockdown organism, containinga ribonucleic acid according to any of the aspects of the presentinvention.

In accordance with a ninth aspect of the present invention there hasbeen provided a composition containing a ribonucleic acid according toany of the aspects of the present invention.

In accordance with a tenth aspect of the present invention there hasbeen provided a pharmaceutical composition containing a ribonucleic acidaccording to any of the aspects of the present invention, and apharmaceutically acceptable carrier.

In accordance with an eleventh aspect of the present invention there hasbeen provided a method for inhibiting the expression of a target gene ina cell or derivative thereof comprising introducing a ribonucleic acidaccording to any of the aspects of the present invention into a cell inan amount sufficient to inhibit expression of the target gene, whereinthe target gene is the target gene of a ribonucleic acid according toany of the aspects of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic illustration defining the terminology as usedherein. The upper of the two strands is the first strand and theantisense strand of the targeted nucleic acid such as mRNA. The secondstrand is the one which essentially corresponds in its sequence to thetargeted nucleic acid and thus forms the sense strand. Both, the firststrand and second strand form a double-stranded structure, typicallythrough Watson Crick base pairing.

FIG. 2 illustrates some embodiments of the ribonucleic acid molecules ofthe present invention with patterns of modified and unmodified groups ofnucleotides which are also referred to herein as a pattern ofmodification. The modified groups of nucleotides are also referred toherein as a group of modified nucleotides. The unmodified nucleotides orunmodified groups of nucleotides referred to as flanking group(s) ofnucleotides herein, as used herein may also have one or several of themodification(s) as disclosed herein which, however, is/are differentfrom the modification of the nucleotides forming the group(s) ofmodified nucleotides. In FIG. 2A the modified and unmodified groups ofnucleotides, i.e. the groups of modified nucleotides and the flankinggroups of nucleotides on both the first stretch and the second stretchare located on corresponding parts of the stretches and are thus alignedto each other (groups of modified nucleotides on the first strandaligned with groups of modified nucleotides on the second strand andflanking groups of nucleotides on the first strand aligned with flankinggroup of nucleotides on the second strand), whereas in FIG. 2B thepattern realised on the first strand is also realised on the secondstrand, however, with a phase shift such that the modified group ofnucleotides of the first stretch is base pairing with an unmodifiedgroup of nucleotides of the second stretch and vice versa so that agroup of modified nucleotides on the first strand aligns with a flankinggroup of nucleotides on the second strand. In FIG. 2C a furtherpossibility of arranging the modified and unmodified groups ofnucleotides is realised. It is also within the present invention thatthe pattern of the first stretch is independent from the pattern of thesecond stretch and that both patterns partially overlap in terms ofrelative position to each other in the double-stranded structure definedby base pairing. In a further embodiment the extent of this overlappingcan vary over the length of the stretch(es) and strand(s), respectively.

FIGS. 3A-3C show the result of a knockdown experiment using RNAimolecules with different end protection groups. More particularly FIG.3A shows that the various forms of end protected RNAi molecules arefunctional on the knockdown of PTEN mRNA.

FIG. 3B (SEQ ID NOs: 1-8, respectively in order of appearance) shows thesequence of the different RNAi molecules used in the experiment theresult of which is depicted in FIG. 3A. FIG. 3C shows the result of animmunoblot analysis of PTEN protein after treatment with modified RNAimolecules in comparison to PTEN specific antisense constructs.

FIGS. 4A-4B show that the 3′ overhang of RNAi molecules is not importantfor RNA interference. More particularly, FIG. 4A shows a dose responsecurve of different RNAi molecules and FIG. 4B (SEQ ID NOs: 9-20,respectively in order of appearance) shows the sequence of the RNAimolecules used in the experiment the result of which is shown in FIG.4A.

FIGS. 5A-5B show that duplex length of the RNAi molecules has to be atleast 18-19 nucleotides. More particularly, FIG. 5B (SEQ ID NOs: 21-28,respectively in order of appearance) shows the sequence of the PTENspecific RNAi molecules used in the experiment the result of which isdepicted in FIG. 5A as dose response curve.

FIGS. 6A-6B show that four terminal mismatched nucleotides in RNAimolecules with a length of 19 nucleotides are still functional inmediating Akt1 knockdown. More particularly, FIG. 6B (SEQ ID NOs: 29-36,respectively in order of appearance) shows the sequence of the RNAimolecules used in the experiment the result of which is depicted in FIG.6A.

FIGS. 7A-7C show further results on duplex length requirements andtolerance for mutation in siRNAs. More particularly, FIG. 7A (SEQ IDNOs: 37-52, respectively in order of appearance) shows the variousconstructs used (left panel) and the respective impact on inhibition ofAkt1 mRNA expression in HeLa cells relative to the expression of p110αused in the indicated amounts of siRNA molecules (right panel). Thenucleotide changes in the mismatch siRNA molecules are indicated byarrows; the 3′ deoxyribonucleotides, if any, are indicated in capitalletters. FIG. 7B (SEQ ID NOs: 53-62, respectively in order ofappearance) shows the various PTEN specific siRNAs (left panel), theinhibition of PTEN mRNA expression in HeLA cells expressed as ratioPTEN/p110α, at various amounts of siRNA (middle panel) and FIG. 7C aWestern Blot analysis depicting the inhibition of PTEN proteinexpression using PTEN specific siRNA (30 nM) and respective mismatchsiRNA after 48 and 96 hours, respectively, with p100α being used asloading control.

FIGS. 8A-8B show the result of studies on the stability in serumconferred to RNAi molecules by 2′-O-methylation and that endmodifications have no beneficial effects on RNAi stability. Moreparticularly, FIG. 8A shows the result of a gel electrophoresis of thevarious RNAi molecules depicted in FIG. 8B (SEQ ID NOs: 9, 10, 63-68,13, 14, 17 and 69-71, respectively in order of appearance) being subjectto incubation with fetal calf serum.

FIGS. 9A-9C show that an amino end modification results in loss ofactivity. FIG. 9B (SEQ ID NOs: 72, 77, 73, 77, 74, 77, 75, 77, 78, 77,76, 77, 80, 77, 78, and 79, respectively in order of appearance) showsthe particular RNAi molecules used in the experiments the result ofwhich is shown in FIG. 9A expressed as PTEN/p110α expression levelratio. FIG. 9C shows the design principles which may be deduced from theresults depicted in FIG. 9A. As used in FIG. 9C the term functionalmeans functionally active in the particular assay system as described inthe example and “not functional” means not functionally active in saidsystem.

FIGS. 10A-10C show that 2′-O-Alkyl (methyl) modifications stabilize RNAimolecules but also result in reduction of their activity. Moreparticularly, FIG. 10C shows the sequence of the RNAi molecules used inthe experiment the result of which is depicted as a dose response curvein FIG. 10A. FIG. 10B shows the result of a gel electrophoresis of thevarious RNAi molecules depicted in FIG. 10C (SEQ ID NOs: 13, 14, 81-90,80, 90, 89, 14, 87 and 90, respectively in order of appearance) beingsubject to a two hour incubation in fetal calf serum.

FIGS. 11A-11C show the result of an experiment on the efficacy of RNAimolecules with blocks of 2′-O-methyl modifications with FIG. 11Agraphically depicting the results of said experiments as a dose responsecurve and with FIG. 11C showing the sequences of the particular RNAimolecules used in said experiments. FIG. 11B shows the result of a gelelectrophoresis of the various RNAi molecules depicted in FIG. 11C (SEQID NOs: 91-97, 90, 98-100 and 90, respectively in order of appearance)being subject to a two hour incubation in fetal calf serum.

FIGS. 12A-12D show that alternating 2′-O-methyl modification result inactivity of the modified RNAi molecules compared to unmodified forms.More particularly, FIG. 12B (SEQ ID NOs: 101-118, respectively in orderof appearance) shows the sequence of the RNAi molecules used in thisexperiment the result of which is depicted in FIG. 12A. FIG. 12C showsthe stability of said RNAi molecules following incubation in serum fortwo hours, whereas FIG. 12D shows an immunoblot for PTEN protein uponapplication of different RNAi molecules to HeLa cells. As may be takentherefrom RNAi molecules with alternating modifications are stabilizedagainst endonuclease degradation and active in mediating a PTEN proteinknock down.

FIG. 13 shows the result of a Western Blot analysis to determine thetime course of PTEN protein knock down. Cells were continuouslytransfected with 2′-O-Methyl modified versus unmodified RNAi moleculesusing cationic lipids for 72 h. Protein lysates were prepared andanalysed by immunoblot after 48 and 120 h. For the 96 h and 120 htimepoints the cells were split, replated and incubated in the absenceof RNAi molecules for an additional 24 and 48 h.

FIG. 14 shows a Western Blot depicting the protein knock down of PTENbeing persistent using alternating modified RNAi molecules versusunmodified RNAi molecules. Transfections were performed for only 5 h andnew medium without transfection reagents was added. Lysates wereanalysed by immunoblot 72 h and 96 h post transfection with theindicated RNAi molecules.

FIGS. 15A-15D show that siRNA molecules with distinct 2′-O-methylribonucleotides modifications show increased stability in serum andmediate protein knock-down in HeLa cells. More particularly, FIG. 15A(SEQ ID NOs: 119-146, respectively in order of appearance) indicates thevarious siRNA molecule constructs used (left panel), whereby 2′-O-methylribonucleotides modifications are underlined and indicated by boldletters in the sequence. Inhibition of PTEN mRNA expression in HeLacells transfected with the indicated amounts of modified siRNA moleculesis expressed as ratio PTEN/p110α and indicated on the right panel. FIG.15B (SEQ ID NOs: 119, 120, 123, 124, 135-138, 141, 142, 147-152, 145 and146, respectively in order of appearance) shows on the left panel thevarious siRNA constructs used and on the right panel a PAA gelelectrophoresis of modified and unmodified siRNA molecules afterincubation in serum; the various constructs with 2′-O-methylribonucleotides are indicated by underlining and bold printing. FIG. 15Cshows an SDS-PAGE based immunoblot illustrating the inhibition of PTENprotein expression using various of the siRNA constructs (30 nM) asdepicted in FIGS. 15A and 15B, respectively. Again, p110α is used asloading control. Finally, FIG. 15D is an immunoblot indicating aprolonged protein knock-down, i.e. the inhibition of PTEN proteinexpression, upon administration of siRNA molecules (30 nM) with distinct2′-O-methylribonucleotides modifications after 48 and 128 hours. As inFIG. 15C, p110α is used as loading control.

FIGS. 16A-16C show that siRNA molecules with distinct2′-O-methylribonucleotides modifications which are specific for Akt1 andp110β mRNA show increased stability in serum and mediate proteinknock-down in HeLa cells. More particularly, FIG. 16A (SEQ ID NOs:153-164, respectively in order of appearance) indicates on the leftpanel the various constructs used whereby again2′-O-methylribonucleotides are underlined and printed in bold. Theintegrity of the indicated siRNA molecules after incubation in serum isshown in the right panel. FIG. 16B shows an immunoblot of Akt1, Akt2 andAkt phosphorylation and p110 being used as a loading control upontransfection of the cells with the indicated siRNAs (30 mM). FIG. 16C(SEQ ID NOs. 165-174, respectively in order of appearance) shows variousp110β specific siRNA constructs (left panel) with the 2′-O-methylmodifications being underlined and printed in bold, and the result of animmunoblot analysis (right panel) of the inhibition of thephosphorylation of the downstream kinase Akt1 by said siRNA constructs.p110α has been used as a loading control.

FIGS. 17A-17B show the efficacy of various RNAi molecules with hairpinstructures as dose response curve while FIG. 17B shows the structure ofthe RNAi molecules the result of which is depicted in FIG. 17A.Synthetic siRNAs with different loops are functional in reducing thep110β, Akt1 and Akt 2 expression. (14A) Inhibition of p110β mRNAexpression in siRNA transfected HeLa cells. Samples were analyzed inparallel for the level of p110β mRNA expression 24 h after transfectionof the indicated siRNAs. The transfected bimolecular siRNAs (21 mer with3′ TT overhangs, molecule 1AB) or the monomolecular siRNAs with loopstructures are schematically shown. Note that the position of the loops(HIV derived pA-loop; (A)₁₂-loop) (SEQ ID NO: 175) relative to theantisense sequence is reversed in 3A, 4A relative to 3B, 4B. The 2ABsiRNA molecule contains 6 mismatches in the 21 mer duplex and serves asa negative control together with the untreated sample. RNA was preparedand subjected to real time RT-PCR (Taqman) analysis. p110β mRNA levelsare shown relative to the mRNA levels of p110α, which serve as aninternal reference. Each bar represents triplicate transfections(±standard deviation). HeLa cells were transfected at 50% confluency(2500 cells per 96 well) with siRNAs at the indicated concentrations ingrowth medium.

FIGS. 18A-18C show the efficacy of various RNAi molecules withintermolecular and intramolecular loop structures as dose responsecurves. (18A) (SEQ ID NO: 175) Inhibition of Akt1 mRNA expression insiRNA transfected HeLa cells. Samples were analysed in parallel for thelevel of Akt1 and Akt2 mRNA expression 24 h after transfection of theindicated siRNAs. The different loops (A-loops; GAGA-loop and apolyethyleneglycol (PEG)-linker) and their putative secondary structureare shown schematically. The siRNA molecule 9A is specific for Akt2 andserves as a negative control. Note that 10A and 10B do not containself-complementary sequences and are transfected in combination. Akt1mRNA levels are shown relative to the mRNA levels of p110β, which servedas internal control. (18B) (SEQ ID NO: 175) Inhibition of Akt2 mRNAexpression in HeLa cells transfected with the indicated siRNA molecules.Akt2 mRNA levels are shown relative to the mRNA levels of p110β. TheAkt1 specific molecule 7A serves here as a negative control.

FIG. 18C shows a Western Blot analysis on Akt protein depicting thefunctionality of synthetic siRNAs with different loops in specificallyreducing the Akt1 and Akt 2 expression. Inhibition of Akt1 and Akt2protein expression were analysed by immunoblot. The cells were harvested48 h after transfection of the indicated hairpin siRNAs (20 nM). Cellextracts were separated by SDS-PAGE and analysed by immunoblotting usinganti-p110 antibody, anti Akt 1/2. Similar results were obtained with anantibody specific for the phosphorylated form of Akt1. The positions ofp110α, another catalytic subunit of PI 3-kinase, which was used as aloading control, and of Akt1, Akt2 and phosphorylated Akt (P*-Akt) areindicated on the left.

FIGS. 19A-19B show an NH₂ modification, also referred to herein as aminomodification, which may be present at either the 3′-OH terminalnucleotide or the 5′ terminal nucleotide. The amino group is attached tothe phosphate which in turn is attached to the OH group of the sugarmoiety, through an alkyl group comprising an alkyl chain of 1 to 8,preferably 6 C atoms, whereby the second C atom close to the phosphategroup has a CH₂OH group attached thereto. As an alternative the linkermay be formed by an ether whereby the ether is comprised of two alcoholswhereby one alcohol is an amino alcohol and the other is a dialcoholwith one alcohol group involved in the formation of the ether group andthe other one being an OH group located at either of the C atoms,preferably at the second C atom relative to the phosphate group.

DETAILED DESCRIPTION

The present inventors have surprisingly found that small interferingRNAs can be designed that are both highly specific and active as well asstable under the reaction conditions typically encountered in biologicalsystems such as biochemical assays or cellular environments. The variousinterfering RNAs previously described by Tuschl et al. (see, forexample, WO01/75164) provide for a length of 21 to 23 nucleotides and amodification at the 3′ end of the double-stranded RNA. The presentinventors found that stability problems of interfering RNA, includingsmall interfering RNA (siRNA) which is generally referred to herein inthe following as RNAi, actually result from attack by endonucleasesrather than exonucleases as previously thought. This surprisingobservation permitted the present inventors to develop the methods andcompositions that are the subject of the present invention.

Structure of RNAi Molecules

The present invention provides new forms of interfering RNA. RNAiconsists of a ribonucleic acid comprising a double-stranded structure,formed by a first strand and a second strand. The first strand comprisesa stretch of contiguous nucleotides (the first stretch of contiguousnucleotides) that is at least partially complementary to a targetnucleic acid. The second strand also comprises a stretch of contiguousnucleotides where the second stretch is at least partially identical toa target nucleic acid. The very basic structure of this ribonucleic acidis schematically shown in FIG. 1. The first strand and said secondstrand may be hybridized to each other to form a double-strandedstructure. The hybridization typically occurs by Watson Crick basepairing.

The ribonucleic acids of the invention are, however, not necessarilylimited in length to such a double-stranded structure. For example,further nucleotides may be added to each strand and/or to each end ofany of the strands forming the RNAi. Depending on the particularsequence of the first stretch and the second stretch, the hybridizationor base pairing is not necessarily complete or perfect, which means thatthe first stretch and the second stretch are not 100% base paired due tomismatches. One or more mismatches may also be present within theduplex. Such mismatches have no effect on the RNAi activity if placedoutside a stretch of preferably 15, 16 or 17 matching nucleotides. Ifmismatches are placed to yield only 15 or less contiguous matchingnucleotides, the RNAi molecule typically shows a reduced activity indown regulating mRNA for a given target compared to a 17 matchingnucleotide duplex.

The first stretch of contiguous nucleotides of the first strand isessentially complementary to a target nucleic acid, more advantageouslyto a part of the target nucleic acid. The term “complementary” as usedherein preferably means that the nucleotide sequence of the first strandhybridizes to a nucleic acid sequence of a target nucleic acid sequenceor a part thereof. Typically, the target nucleic acid sequence or targetnucleic acid is, in accordance with the mode of action of interferingribonucleic acids, a single stranded RNA, more preferably an mRNA. Suchhybridization occurs most likely through Watson Crick base pairing but,however, is not necessarily limited thereto. The extent to which thefirst strand and more particularly the first stretch of contiguousnucleotides of the first strand is complementary to a target nucleicacid sequence can be as high as 100% and be as little as 80%,advantageously 80-100%, more advantageously 85-100%, most advantageously90-100%. Optimum complementarity seems to be 95-100%. Complementarity inthis sense means that the aforementioned range of nucleotides, such as,e.g., 80%-100%, depending on the particular range, of the nucleotidesare perfect by Watson Crick base pairing.

Complementarity of the Strands

It is shown in one aspect of the present invention that thecomplementarity between the first stretch of nucleotides and the targetRNA has to be 18-19 nucleotides; stretches of as little as 17nucleotides even with two sequence specific overhangs are not functionalin mediating RNAi. Accordingly, in a duplex having a length of 19nucleotides or base pairs a minimum complementarity of 17 nucleotides ornucleotide base pairs is acceptable, allowing for a mismatch of twonucleotides. In the case of a duplex consisting of 20 nucleotides orbase pairs a complementarity of 17 nucleotides or nucleotide base pairsis allowable and functionally active. The same principle applies to aduplex of 21 nucleotides or base pairs with a total of 17 complementarynucleotides or base pairs. The extent of complementarity required for alength of a duplex, i.e. of a double stranded structure, can also bebased on the melting temperature of the complex formed by either thedouble stranded structure as described herein or by the complex of thefirst stretch of the first strand and the target nucleic acid.

The skilled artisan will understand that all of the ribonucleic acids ofthe present invention are suitable to cause or being involved in methodsof RNA mediated interference such as those described, for example, in WO99/32619, WO 00/44895 and WO 01/75164.

Length of the RNAi Molecules

The first strategy by which an interfering ribonucleic acid molecule maybe designed according to the present invention is to have an optimumlength of 18 or 19 nucleotides of the stretch that is complementary tothe target nucleic acid. It is also within the scope of the presentinvention that this optimum length of 18 or 19 nucleotides is the lengthof the double stranded structure in the RNAi used. This lengthrequirement clearly differs from that described in, for example, WO01/75164. It is within the scope of the present invention that anyfurther design, both according to the present invention and aspreviously described by others, can be realised in connection with aninterfering ribonucleic acid having such length characteristics, i.e. alength of 18 or 19 nucleotides.

End Modification of the RNAi Molecules

The second strategy for the design of an interfering ribonucleic acidmolecule is to have a free 5′ hydroxyl group, (also referred to hereinas a free 5′ OH-group) at the terminus of the first strand. A free 5′OH-group means that the most terminal nucleotide forming the firststrand is present and is thus not modified, particularly not by an endmodification. Typically, the terminal 5′-hydroxy group of the secondstrand, respectively, is also present in an unmodified manner. In a morepreferred embodiment, the 3′-end of the first strand and first stretch,respectively, also is unmodified so as to present a free OH-group (alsoreferred to herein as a free 3′OH-group), whereby the design of the 5′terminal nucleotide is the one of any of the embodiments describedabove. Preferably such a free OH-group also is present at the 3′-end ofthe second strand and second stretch, respectively. In other embodimentsof the ribonucleic acid molecules as described previously according tothe present invention the 3′-end of the first strand and first stretch,respectively, and/or the 3′-end of the second strand and second stretch,respectively, may have an end modification at the 3′ end.

As used herein the terms free 5′OH-group and 3′OH-group also indicatethat the respective 5′ and 3′ terminal nucleotides of the polynucleotidepresent an OH-group. Such OH-group may stem from either the sugar moietyof the nucleotide, more preferably from the 5′ position in the case ofthe 5′OH-group and from the 3′ position in the case of the 3′OH-group,or from a phosphate group attached to the sugar moiety of the respectiveterminal nucleotide. The phosphate group may in principle be attached toany OH-group of the sugar moiety of the nucleotide. Preferably, thephosphate group is attached to the 5′OH-group of the sugar moiety in thecase of the free 5′OH-group and/or to the 3′OH-group of the sugar moietyin the case of the free 3′OH-group still providing what is referred toherein as free 5′ or 3′ OH-group.

The term “end modification” as used herein in connection with anystrategy for the design of RNAi or any embodiment of RNAi disclosedherein, means a chemical entity added to the most 5′ or 3′ nucleotide ofthe first and/or second strand. Examples of such end modificationsinclude, but are not limited to, inverted (deoxy) abasics, amino,fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioate,C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl,OCF₃, OCN, O—, S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂, N₃; heterocycloalkyl, heterocycloalkaryl, aminoalkylamino;polyalkylamino or substituted silyl, as, among others, described inEuropean patents EP 0 586 520 B1 or EP 0 618 925 B1.

As used herein, alkyl or any term comprising “alkyl” means any carbonatom chain comprising 1 to 12, preferably 1 to 6 and more, preferably 1to 2 C atoms.

A further end modification is a biotin group, which may preferably beattached to either the most 5′ or the most 3′ nucleotide of the firstand/or second strand, or to both ends. In a more preferred embodimentthe biotin group is coupled to a polypeptide or a protein. It is alsowithin the scope of the present invention that the polypeptide orprotein is attached through any of the end modifications describedherein. The polypeptide or protein may confer further characteristics tothe nucleic acid molecules of the invention. For example, thepolypeptide or protein may act as a ligand to another molecule. If theother molecule is a receptor the receptor's function and activity may beactivated by the binding ligand. The receptor may show aninternalization activity which provides for effective transfection ofthe ligand bound inventive nucleic acid molecules. An example of aligand that may be coupled to the nucleic acid molecules of theinvention is VEGF and the corresponding receptor is the VEGF receptor.

Various possible embodiments of the RNAi of the present invention havingdifferent kinds of end modification(s) are presented in the Table 1below:

TABLE 1 Various embodiments of the interfering ribonucleic acidaccording to the present invention 2^(nd) strand/2nd 1^(st)strand/1^(st) stretch stretch 1.) 5′-end free OH free OH 3′-end free OHfree OH 2.) 5′-end free OH free OH 3′-end end modification endmodification 3.) 5′-end free OH free OH 3′-end free OH end modification4.) 5′-end free OH free OH 3′-end end modification free OH 5.) 5′-endfree OH end modification 3′-end free OH free OH 6.) 5′-end free OH endmodification 3′-end end modification free OH 7.) 5′-end free OH endmodification 3′-end free OH end modification 8.) 5′-end free OH endmodification 3′-end end modification end modification

The various end modifications as disclosed herein are preferably locatedat the ribose moiety of a nucleotide of the ribonucleic acid. Moreparticularly, the end modification may be attached to or replace any ofthe OH-groups of the ribose moiety, including but not limited to the2′OH, 3′OH and 5′OH position, provided that the nucleotide thus modifiedis a terminal nucleotide. Inverted abasics are nucleotides, eitherdeoxyribonucleotides or ribonucleotides which do not have a nucleobasemoiety. This kind of compound is, among others, described in Sternbergeret al. (2002), Antisense. Nucl. Ac. Drug Dev. 12:143.

Any of the aforementioned end modifications may be used in connectionwith the various embodiments of RNAi depicted in Table 1. It isparticularly advantageous to inactivate the sense strand of any of theRNAi forms or embodiments disclosed herein, preferably via an endmodification, and more preferably a 5′ end modification. The advantageof this strategy arises from the inactivation of the sense strand whichcorresponds to the second strand of the ribonucleic acids describedherein, which might otherwise interfere with an unrelatedsingle-stranded RNA in the cell. Thus the expression and moreparticularly the translation pattern of the transcriptome of a cell ismore specifically influenced. This effect is also referred to as anoff-target effect. Referring to Table 1 those embodiments depicted asembodiments 7 and 8 are particularly advantageous in the above sense asthe modification results in an inactivation of the—targetunspecific—part of the RNAi (which is the second strand) thus reducingany unspecific interaction of the second strand with single-stranded RNAin a cellular or similar system where the RNAi according to the presentinvention is going to be used to knock down specific ribonucleic acidsand proteins, respectively.

Blunt-Ended RNAi Molecules

A third strategy provided by the present invention involves aribonucleic acid comprising a double-stranded structure having a firststrand and a second strand, where the first strand comprises a firststretch of contiguous nucleotides and where the first stretch is atleast partially complementary to a target nucleic acid, and the secondstrand comprises a second stretch of contiguous nucleotides where thesecond stretch is at least partially identical to a target nucleic acid,where the double-stranded structure is blunt-ended. As used herein theterm double-stranded structure also is referred to as duplex. Thisdesign of RNAi clearly differs from, e.g., that described by Tuschl etal. in WO 01/75164, which contains a 3′-overhang. As used herein theterm overhang refers to a double-stranded structure where at least oneend of one strand is longer than the corresponding end of the otherstrand (“the counter strand”) forming the double-stranded structure.Preferably, the first stretch is identical to the first strand and thesecond stretch is identical to the second strand.

It also is advantageous to employ a combination of the design principlesdescribed above, i.e. to prepare RNAi molecules that are blunt-ended andthat have an end modification of either the first or the second strandor both. In other words, it is within the scope of the present inventionto have blunt-ended RNAi carrying any end modification scheme asdepicted in Table 1.

RNAi Molecules Having a 5′ Overhang

The fourth strategy of the present invention is to have an overhang atthe 5′-end of the ribonucleic acid. More particularly, such overhang mayin principle be present at either or both the first strand and secondstrand of the ribonucleic acid according to the present invention. Thelength of the overhang may be as little as one nucleotide and as long as2 to 8 nucleotides, preferably 2, 4, 6 or 8 nucleotides. It is withinthe present invention that the 5′ overhang may be located on the firststrand and/or the second strand of the ribonucleic acid. Thenucleotide(s) forming the overhang may be (a) deoxyribonucleotide(s),(a) ribonucleotide(s) or a continuation thereof.

The overhang preferably comprises at least one deoxyribonucleotide,whereby said one deoxyribonucleotide is preferably the most 5′-terminalone. It is within the present invention that the 3′-end of therespective counter-strand of the inventive ribonucleic acid does nothave an overhang, more preferably not a deoxyribonucleotide overhang.Here again, any of the inventive ribonucleic acids may comprise an endmodification scheme as outlined in connection with Table 1 and/or an endmodification as outlined herein.

RNAi Molecules Having Modified Nucleotides

A fifth strategy for the design of interfering ribonucleic acids subjectto the present application resides in the formation of a certain patternof modified nucleotides on at least one of the strands and moreparticularly on one of the stretches of contiguous nucleotides of theribonucleic acid(s) according to the present invention. The type ofmodification of the nucleotides may be the same as that discussed inconnection with the other strategies for designing interfering RNAdisclosed herein. Advantageously, the modification is an endmodification as described herein, such as, e.g., inverted abasics,methoxy, or amino and the like at the ribose moiety of at least onenucleotide forming the ribonucleotide acids according to the presentapplication. The modification of the nucleotides may be any form ofmodification described herein, more particularly the kind ofmodification as described herein as end modification, except that theso-called end modification is not necessarily located at terminalnucleotides. Rather the modification may occur at a non-terminalnucleotide. Under such conditions the modification is preferablyattached to the ribose moiety of the—to be—modified nucleotide and evenmore preferably to the 2′ position of the ribose moiety.

Combinations of Design Elements

It is also within the present invention that any ribonucleic aciddesigned according to this strategy may also have the features conferredto a ribonucleic acid according to the present application by any of theother design strategies disclosed herein. Accordingly, the interferingribonucleic acid having a pattern of modified nucleotides may have anend modification, an end modification scheme, may be blunt ended or mayhave a 5′ overhang or any combination of two or more of these elementsor characteristics.

Apart from the aforementioned modifications which may be presentedeither as end modifications or as modification pattern, the ribonucleicacid backbone as such may be further modified by forming different linksbetween the nucleotides. Examples of such different links are described,among others, in EP 0 586 520 B1 and EP 0 618 925 B1. Of particularinterest here are internal modification(s) of the ribonucleic acidbackbone which have been shown to confer higher nuclease resistance ofribooligonucleotides. In a preferred embodiment the modification of themodified nucleotide is a methylation of the 2′-OH-group of the ribosemoiety of the nucleotide to form a methoxy group.

In a preferred embodiment, both strands, and more particularly both thefirst stretch and the second stretch show this kind of modification ofthe nucleotides forming said strands and stretches, respectively.However, it is also within the present invention that either the firststrand and first stretch, respectively, or the second strand and secondstretch, respectively, show this particular pattern of modification ofthe nucleotides. As used herein, the term group of modified nucleotidesor flanking group of nucleotides may comprise or represent one or morenucleotides.

A pattern of modification of the nucleotides in a contiguous stretch ofnucleotides may be realised such that the modification is containedwithin a single nucleotide or group of nucleotides that are covalentlylinked to each other via standard phosphodiester bonds or, at leastpartially, through phosphorothioate bonds. In the event that such amodified nucleotide or group of modified nucleotides does not form the5′-end or 3′-end of the stretch, a flanking nucleotide or group ofnucleotides is arrayed on both sides of the modified nucleotide orgroup, where the flanking nucleotide or group either is unmodified ordoes not have the same modification of the preceding nucleotide or groupof nucleotides. The flanking nucleotide or group of nucleotides may,however, have a different modification. This sequence of modifiednucleotide or group of modified nucleotides, respectively, andunmodified or differently modified nucleotide or group of unmodified ordifferently modified nucleotides may be repeated one or more times.Preferably, the sequence is repeated more than one time. The pattern ofmodification is discussed in more detail below, generally referring to agroup of modified nucleotides or a group of unmodified nucleotideswhereby each of said group may actually comprise as little as a singlenucleotide. The term “unmodified nucleotide” as used herein means eithernot having any of the aforementioned modifications at the nucleotideforming the respective nucleotide or group of nucleotides, or having amodification which is different from the one of the modified nucleotideand group of nucleotides, respectively.

It is also within the present invention that the modification of theunmodified nucleotide(s) wherein such unmodified nucleotide(s) is/areactually modified in a way different from the modification of themodified nucleotide(s), can be the same or even different for thevarious nucleotides forming said unmodified nucleotides or for thevarious flanking groups of nucleotides. That is, a group of modifiednucleotides may contain two or more different modified nucleotideswithin a single group.

The pattern of modified and unmodified nucleotides may be such that the5′-terminal nucleotide of the strand or of the stretch starts with amodified group of nucleotides or starts with an unmodified group ofnucleotides. However, in an alternative embodiment it is also possiblethat the 5′-terminal nucleotide is formed by an unmodified group ofnucleotides.

This kind of pattern may be realised either on the first stretch or thesecond stretch of the interfering RNA or on both. A 5′ phosphate on thetarget-complementary strand of the siRNA duplex is required for siRNAfunction, suggesting that cells check the authenticity of siRNAs througha free 5′ OH (which can be phosphorylated) and allow only such bona fidesiRNAs to direct target RNA destruction (Nykanen, et al. (2001), Cell107, 309-21).

Preferably, the first stretch shows a pattern of modified and unmodifiedgroups of nucleotides, i.e. of group(s) of modified nucleotides andflanking group(s) of nucleotides, whereas the second stretch does notshow this kind of pattern. This may be useful insofar as the firststretch is actually the more important one for the target-specificdegradation process underlying the interference phenomenon of RNA sothat for specificity reasons the second stretch can be chemicallymodified so it is not functional in mediating RNA interference.

However, it is also within the present invention that both the firststretch and the second stretch have this kind of pattern. Preferably,the pattern of modification and non-modification is the same for boththe first stretch and the second stretch.

In a particular embodiment, the modified nucleotides or groups ofmodified nucleotides of one strand of the molecule are complementary inposition to the modified nucleotides or groups of nucleotides of theother strand. This possibility is schematically depicted in FIG. 2A. Inan alternative embodiment, there is a phase shift between the patternsof modification of the first stretch and first strand, respectively,relative to the pattern of modification of the second stretch and secondstrand, respectively. Preferably, the shift is such that the modifiedgroup of nucleotides of the first strand corresponds to the unmodifiedgroup of nucleotides of the second strand and vice versa. Thispossibility is illustrated schematically in FIG. 2B. In anotherembodiment, there is a partial shift of the pattern of modification sothat the modified groups overlap as illustrated in FIG. 2C. The groupsof modified nucleotides in any given strand may optionally be the samelength, but may be of different lengths. Similarly, the groups ofunmodified nucleotides in any given strand may optionally be the samelength, or of different lengths.

In a preferred embodiment the second (penultimate) nucleotide at theterminus of the strand and stretch, respectively, is an unmodifiednucleotide or the beginning group of unmodified nucleotides. Preferably,this unmodified nucleotide or unmodified group of nucleotides is locatedat the 5′-end of the first and second strand, respectively, and evenmore preferably at the terminus of the first strand. In a furtherpreferred embodiment the unmodified nucleotide or unmodified group ofnucleotides is located at the 5′-end of the first strand and firststretch, respectively. In a preferred embodiment the pattern consists ofalternating single modified and unmodified nucleotides.

In a particular embodiment of this aspect of the present invention theinterfering ribonucleic acid subject comprises two strands, whereby a2′-O-methyl modified nucleotide and a non-modified nucleotide,preferably a nucleotide which is not 2′-O-methyl modified, areincorporated on both strands in an alternating fashion, resulting in astrand having a pattern MOMOMOMOM etc. where M is a 2′-O-methylnucleotide and O is a non-modified nucleotide. The same sequence of2′-O-methyl modification and non-modification exists on the secondstrand, and preferably there is a phase shift between the two strandssuch that the 2′-O-methyl modified nucleotide on the first strand basepairs with a non-modified nucleotide(s) on the second strand and viceversa. This particular arrangement. i.e. base pairing of 2′-O-methylmodified and non-modified nucleotide(s) on both strands is particularlypreferred in case of short interfering ribonucleic acids, i.e. shortbase paired double-stranded ribonucleic acids because it is assumed,although the present inventors do not wish to be bound by that theory,that a certain repulsion exists between two base-pairing 2′-O-methylmodified nucleotides which would destabilise a duplex, and particularlyshort duplexes, containing such pairings.

In a “phase shifted” arrangement of this nature, it is preferred if theantisense strand starts with a 2′-O-methyl modified nucleotide at the 5′end whereby consequently the second nucleotide is non-modified, thethird, fifth, seventh and so on nucleotides are thus again 2′-O-methylmodified whereas the second, fourth, sixth, eighth and the likenucleotides are non-modified nucleotides. Again, not wishing to be boundby any theory, it seems that a particular importance may be ascribed tothe second, and optionally fourth, sixth, eighth and/or similarposition(s) at the 5′ terminal end of the antisense strand which shouldnot comprise any modification, whereas the most 5′ terminal nucleotide,i.e. the first 5′ terminal nucleotide of the antisense strand mayexhibit such modification with any uneven positions such as first,optionally third, fifth and similar position(s) at the antisense strandmay be modified. In further embodiments the modification andnon-modification, respectively, of the modified and non-modifiednucleotide(s), respectively, may be any modification as describedherein.

Although not limited thereto, the double-stranded structure of theinventive ribonucleic acid, which is also referred to as duplex, isformed by the first strand and second strand, respectively, or the firstand second stretch of contiguous nucleotides. The length of the firststretch and second stretch, respectively, is typically about 15 to about23, preferably 17 to 21, and more preferably 18 or 19 bases. A length ofless than 30 nucleotides, preferably less than 21 nucleotides does notinduce an interferon response in any biological system which is capableof showing RNA interference and an interferon response. This apparentlyis because a given cell experiences profound physiological changes whendouble-stranded RNA longer than 30 base pairs binds and activates theprotein kinase PKR and 2′,5′-oligoadenylate synthetase. Activated PKRstalls translation via phosphorylation of eIF2a, activated 2′,5′-AScauses mRNA degradation. These effects are not desired in targetvalidation and animal models because they override the effect of thetarget specific knockdown on the phenotype.

RNAi Oligonucleotides with Loop Structures

In a sixth strategy for designing interfering ribonucleic acids of thepresent invention, the ribonucleic acid comprises a double-strandedstructure where the double-stranded structure has a first strandcomprising a first stretch of contiguous nucleotides that is at leastpartially complementary to a target nucleic acid, and a second strandcomprising a second stretch of contiguous nucleotides that is at leastpartially identical to a target nucleic acid, and where one terminus ofthe first strand and one terminus of the second strand are linked by aloop structure.

In one embodiment the loop structure is comprised of a non-nucleic acidpolymer. Suitable non-nucleic acid polymers include polyethylene glycolor similar polymers. The polymer may be chosen in such a manner that thetwo linked strands may hybridize to each other. This requires that thepolymer has to have a certain molecular “hinge” structure or molecularflexibility to allow the bending of the molecule so as to allow thatboth stretches get in close contact and in a three-dimensionalorientation which permits hybridization. In principle any molecule whichcomplies with this requirement may be used in connection with thepresent invention. For example, amino acid based molecules may also beused. Such amino acid based molecules may be either homopolymers orhetereopolymers. A useful example is a homopolymer consisting of sevenglycine residues which allows the generation of a hinge as required tobring the two stretches to hybridize in the close proximity as needed. Asuitable glycine based hinge is described, e.g., in Guan K. L. and DixonJ. E. (1991), Anal. Biochem. 192, 262 In another embodiment the hingemay be formed by crown ethers of a type that is known in the art.

In an alternative embodiment the loop is comprised of a nucleic acidmoiety. In the context of these loops, LNA as described in Elayadi andCorey (2001) Curr. Opin. Investig. Drugs. 2(4):558-61 and Orum andWengel (2001) Curr. Opin. Mol. Ther. 3(3):239-43, and PNA are regardedas nucleic acids and may also be used as loop forming polymers.Basically, the 5′-terminus of the first strand may be linked to the3′-terminus of the second strand. As an alternative, the 3′-end of thefirst strand may be linked to the 5′-terminus of the second strand. Thenucleotide sequence forming the loop structure is, in general, notcritical. However, the length of the nucleotide sequence forming suchloop seems to be critical for steric reasons. Accordingly, a minimumlength of four nucleotides appears to be appropriate to form therequired loop structure. In principle, the maximum number of nucleotidesforming the hinge or the link between both stretches to be hybridized isnot limited. However, the longer a polynucleotide is, the more likelysecondary and tertiary structures are formed and this can affect therequired orientation of the stretches that must form the hybridisedstructure. Preferably, a maximum number of nucleotides forming the hingeis about 12 nucleotides. It is within the scope of the present inventionthat any of the designs described above may be combined with this sixthstrategy, i.e. by linking the two strands covalently in a manner thatback folding (loop) can occur through a loop structure or similarstructure.

The present inventors surprisingly have found that if the loop is placed3′ of the antisense strand, i.e. the first strand of the ribonucleicacid(s) according to the present invention, the resulting RNAi moleculeshave higher activity than molecules where the loop is placed 5′ of theantisense strand. This result is contrary to the conventional wisdomthat the orientation of the loop is irrelevant, based on the assumptionthat any RNAi, is subject to a processing during which non-loop linkedRNAi is generated. If this was the case, however, the clearly observedincreased activity of those structures having the loop placed 3′ of theantisense could not be explained. Accordingly, a preferred arrangementin 5′→3′ direction of this kind of small interfering RNAi is secondstrand—loop—first strand.

Expression of RNAi from a Vector

The respective constructs may be incorporated into suitable vectorsystems. Preferably the vector comprises a promoter for the expressionof RNAi. Suitable promoters include pol III and the U6, H1, 7SKpromoters as described in Good et al. (1997) Gene Ther, 4, 45-54.

Use of RNAi for Gene Knockdown and Target Validation

The ribonucleic acid molecules according to the present invention havegeneral applicability and permit knockdown or knockout of any desiredcoding nucleotide such as an mRNA, i.e. the expression of any geneproducing an RNA may be modified. A particular application is the use ofthe inventive ribonucleic acid for target validation. As used herein,target validation means a process that involves determining whether aDNA, RNA, or protein molecule is directly involved in a biologicalprocess. Preferably the biological process is causally involved in adisease or non-standard condition and the gene under study is thereforea suitable target for development of a new therapeutic compound.

Sequence homology studies have successfully classified genes into targetfamilies. The enormous task of deciphering which of these targets arekey players in diseases and which should be used for subsequent drugdevelopment needs to be addressed in an efficient manner. Therefore, theknockdown of gene expression should be reduced by 50-100%, preferably by90% to see significant effects on the phenotype. In other casesdepending on the gene, a knockdown of as little as 20% might besufficient to yield a phenotype. A phenotype will be defined bycomparison of cells containing functional RNAi molecules with cellscontaining non functional RNAi molecules. This will ensure a significantreadout even under conditions where the protein function is inhibitedonly partially. Generally there is no linear correlation between thedegree of mRNA reduction and the extent of the change in phenotype. Forsome proteins a reduction of about 20% of the protein is sufficient tocreate a change in the phenotype whereas in case of other genes andmRNA, respectively, as little as 5 to 10% remaining protein issufficient to maintain an observed phenotype.

A further use of the ribonucleic acid molecules according to the presentinvention is its use for the manufacture of a medicament or its use as amedicament. Such a medicament may either be used for the treatmentand/or prevention of diseases or conditions such as any type of cancerwhere a gene and its product have been linked to the onset, cause orprogression of this disease. Additionally, such a medicament may be usedto treat diseases where the presence or overexpression of a gene productcauses a pathological phenotype. In a preferred embodiment the diseaseis characterised by a gain of function and may be remedied throughapplication or administration of the corresponding, biologically activeRNAi. Diseases or conditions which may be treated by the medicamentcomprising a ribonucleic acid as disclosed herein, may be selected fromthe group comprising cancer, heart diseases, metabolic diseases,dermatological diseases, inflammatory diseases, immune system disordersand autoimmune disorders. The various forms of cancer include, but arenot limited to, solid tumors and tumors of the hematopoietic system,such as glioblastoma, prostate cancer, breast cancer, lung cancer, livercancer, pancreatic cancer and leukaemia. Metabolic diseases include, butare not limited to, obesity and diabetes. Dermatological diseasesinclude, but are not limited to, psoriasis.

In another aspect the ribonucleic acid molecules according to thepresent invention may be used as diagnostics, preferably for thosediseases as specified in connection with the above-mentioned diseasesand conditions. Such diagnosis could be based on the observation thatupon applying the ribonucleic acid molecules according to the presentinvention to a sample which preferably contains cells, a change in theexpression pattern of the sample occurs. Preferably such samplecomprises cells from a subject from whom it is assumed that it mayexhibit said disease or condition to be treated or a predispositiontherefor.

A further application of the nucleic acids according to the presentinvention resides in their use in the screening of pharmaceuticallyactive compounds and/or lead optimization. The latter is done such as tomonitor or determine the effect of candidate drugs such as smallmolecules and compare the effect created by said candidate drugs withthe effect observed upon administering specific RNAi designed on thebasis of the principles disclosed herein. In doing so candidate drugshaving off-target effects may be eliminated from the screening processwhereas those candidate drugs which create a similar or identicalphenotype are deemed as highly relevant lead compound or may even be apharmaceutically active compound themselves. In this approach, thehighly specific RNAi molecules act as a gold standard against whichcandidate drugs are measured.

In a further aspect the invention is related to a cell, preferably aknockdown cell, which contains a ribonucleic acid as disclosed herein.Such a cell preferably is either isolated or contained in a tissue oreven organ which again preferably is not contained in an organism.However, the cell may also be contained in an organism. The cell ispreferably a cell which is involved in the disease or condition which isto be treated by the ribonucleic acids of the invention. This kind ofknock-down cell may be used to generate an expression profile based on,e.g., mRNA or protein, in order to elucidate functional relationship andto determine downstream targets.

In a further aspect the invention is related to an organism containing aribonucleic acid as disclosed herein. Preferably such organism is avertebrate organism and more preferably the vertebrate organism is amammal. A mammal as used herein is, among others and not limitedthereto, an ape, a dog, a cat, a goat, a sheep, a pig, a guinea pig, arabbit, a mouse, a rat and a human being.

In a still further aspect the present invention provides compositionscontaining one or more ribonucleic acids according to the presentinvention. Preferably such composition comprises negative and positivecontrols either in combination with the effective ribonucleic acid orseparated therefrom. Such composition may further comprise a solvent,preferably a buffer.

In a further aspect the present invention is related to a pharmaceuticalcomposition containing a ribonucleic acid according to the presentinvention and a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers are known in the art and comprise, among others,diluent, buffers and the like. Suitable carriers are described, forexample, in Remington's Pharmaceutical Sciences (18th edition, 1990,Mack Publishing Co.). The pharmaceutical composition may compriseadditional pharmaceutically active compounds. In cases where the diseaseor condition to be treated using the ribonucleic acid molecules of thepresent invention is the same or related disease of condition as thattreated by the additional pharmaceutically active compound(s) then thedifferent mode of action of the ribonucleic acid molecules and theadditional compounds will produce synergistic effects.

The invention is now further illustrated by reference to the figures andexamples from which further features, embodiments and advantages of thepresent invention may be taken.

Example 1: Dose Response of Synthetic Duplex RNAi Molecules

In this example the impact of NH₂ end protection groups on the activityof duplex RNAi molecules was investigated. Synthetic siRNAs werepurchased from Biospring (Frankfurt, Germany). The ribooligonucleotideswere resuspended in RNase free TE to a final concentration of 50 μM. Inthe case of bimolecular siRNA molecules equal aliquots (100 μM) werecombined to a final concentration of 50 μM. For the formation ofintramolecular duplexes the siRNAs were incubated at 50° C. for 2 min inannealing buffer (25 mM NaCl; 5 mM MgCl₂) and were cooled down to RT.Transfections were carried out in 96 well or 10-cm plates (at 30% to 50%confluency) by using various cationic lipids such as Oligofectamine,Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals,Inc., Boulder, Colo.), or FuGene 6 (Roche) according to themanufacturer's instructions. RNAi molecules were transfected by addingpre-formed 5× concentrated complex of annealed RNAi and lipid inserum-free medium to cells in complete medium. Prior to transfection2500 HeLa cells were plated per well 15-18 hours before transfection forthe 96 well format.

The total transfection volume was 100 μl for cells plated in 96-wellsand 10 ml for cells in 10 cm plates. The final lipid concentration was0.8 to 1.2 μg/ml depending on cell density; the RNAi concentration isindicated in each experiment.

Complex formation was allowed to take place for 30 min at 37° C.Complexes were added to cells to yield a final 1× concentration of bothlipid and RNAi. Depending on the analysis performed followingtransfection, cells were lysed using a standard cell lysis buffer forprotein extraction (Klippel, A., Escobedo, J. A., Hirano, M. andWilliams, L. T. (1994). Mol Cell Biol 14, 2675-2685) or a denaturingbuffer for RNA isolation according to the RNA isolation kit (Invitek,Berlin (Germany) 24 to 48 hours post transfection for RNA analysis and48 to 72 hours post transfection for protein analysis by Western Blot.

Determination of the Relative Amounts of RNA Levels by Taqman Analysis:

24 h post transfection the RNA of cells transfected in 96-wells wasisolated and purified using the Invisorb RNA HTS 96 kit (InVitek GmbH,Berlin). Inhibition of PTEN mRNA expression was detected by real timeRT-PCR (Taqman) analysis using 300 nM PTEN 5′ primerCACCGCCAAATTTAACTGCAGA (SEQ ID NO: 176), 300 nM PTEN 3′ primerAAGGGTTTGATAAGTTCTAGCTGT (SEQ ID NO: 177) and 100 nM of the PTEN Taqmanprobe Fam-TGCACAGTATCCTTTTGAAGACCATAACCCA-Tamra (SEQ ID NO: 178) incombination with 40 nM β-actin 5′ primer GTTTGAGACCTTCAACACCCCA (SEQ IDNO: 179), 40 nM β-actin 3′ primer GACCAGAGGCATACAGGGACA (SEQ ID NO: 180)and 100 nM of the β-actin Taqman probeVic-CCATGTACGTAGCCATCCAGGCTGTG-Tamra (SEQ ID NO: 181). The Akt primersand probes are determined in Sternberger et al. (Sternberger, supra) andare used according to the manufacturer's instructions (AppliedBiosystem; use of Amplicon Set). Also said primers and probes may bedesigned using the software program Primer Express (Applied Biosystem).The reaction was carried out in 50 μl and assayed on the ABI PRISM 7700Sequence detector (Applied Biosystems) according to the manufacturer'sinstructions under the following conditions: 48° C. for 30 min, 95° C.for 10 min, followed by 40 cycles of 15 sec at 95° C. and 1 min at 60°C.

RNA knockdown was shown by real time RT-PCR analysis of HeLa cellstransfected with 21 nt long siRNA duplex molecules unmodified andmodified with either NH₂ or inverted Abasics groups at the 5′-end at alipid carrier concentration of 1.0 μg/ml. Cell density was 2000cells/well. Modifications on the 3′-end were either RNA overhangs, RNAoverhangs with amino groups or DNA overhangs.

Preparation of cell extracts and immunoblotting. Cells were washed twicewith cold phosphate-buffered saline and lysed at 4° C. in lysis buffercontaining 20 mM Tris (pH 7.5), 137 mM NaCl, 15% (vol/vol) glycerol, 1%(vol/vol) Nonidet P-40 (NP-40), 2 mM phenylmethylsulfonyl fluoride, 10mg aprotinin per ml, 20 mM leupeptin, 2 mM benzamidine, 1 mM sodiumvanadate, 25 mM β-glycerol phosphate, 50 mM NaF and 10 mM NaPPi. Lysateswere cleared by centrifugation at 14,000×g for 5 minutes and aliquots ofthe cell extracts containing equal amounts of protein were analyzed forprotein expression by Western-blotting: Samples were separated bySDS-PAGE and transferred to nitrocellulose-filters (Schleicher &Schuell). Filters were blocked in TBST buffer (10 mM Tris-HCl (pH 7.5),150 mM NaCl, 0.05% (vol/vol) Tween 20, 0.5% (wt/vol) sodium azide)containing 5% (wt/vol) dried milk. The respective antibodies were addedin TBST at appropriate dilutions. Bound antibody was detected usinganti-mouse- or anti-rabbit-conjugated horseradish peroxidase(Transduction Laboratories) in TBST, washed, and developed using theSuperSignal West Dura (Pierce) or ECL (Amersham) chemoluminescencesubstrates (c.f. Sternberger et al. (2002). Antisense. Nucl. Ac. DrugDev. in press.

Antibodies. The murine monoclonal anti-p110 antibody U3A and the murinemonoclonal anti-p85 antibody N7B have been described (Klippel et al.,1994, supra). Rabbit polyclonal anti-Akt and anti-phospho Akt (S473)antibodies were obtained from Cell Signaling Technology. The murinemonoclonal anti-PTEN antibody was from Santa Cruz Biotechnology. ThePTEN 53 specific antisense molecule, i.e. geneBloc, is described inSternberger et al. [Sternberger, supra] having the following sequence(ucuccuuTTGTTTCTGcuaacga) (SEQ ID NO: 182), whereby the nucleotidedepicted in lower case are ribonucleotides whereas the nucleotide incapital letters are deoxyribonucleotide. This antisense molecule is alsoidentical to RNAi 1A without TT.

The results are shown in FIG. 3A and the respective RNAi molecules inFIG. 3B which are directed to the mRNA of PTEN. The nucleotides writtenin lower case letters represent ribonucleotides whereas capital lettersrepresent deoxyribonucleotide. The term NH₂ indicates that the3′-position of the ribonucleotide was modified by an amino group. TheRNAi molecules used in this and other examples disclosed herein are alsoreferred to as small interfering RNA molecules siRNA. It is to be notedthat in any of the figures contained herein the upper strand is theantisense or first strand, whereas the lower strand is the sense orsecond strand of the interfering RNA molecule.

As can be taken from FIG. 3A amino end modifications such as aminomodification and inverted abasics modification of the terminal OH groupof the nucleic acid are as potent as unmodified ends when themodification is located at the 3′ end of the antisense strand (see alsoFIG. 8A; 8B). Therefore chemical modification to stabilize or with otherbeneficial properties (delivery) will be tolerated without activity losswhen located at the 3′ OH, especially when the 3′OH is located on anoverhanging nucleotide.

For the experiment shown in FIG. 3C similar conditions as outlined abovewere used. The first strand and the second strand of the RNAi wereeither modified by a NH₂ group at the 3′-position of the ribose moietyor by an inverted abasic at said positions. The first construct isdesignated as siRNA-NH₂ (3A3B) the second as siRNA-iB (4A4B). Thesequence of both molecules is depicted in FIG. 3B. The term 3A3Bindicates that the interfering ribonucleic acid consists of strand 3A asthe antisense strand and strand 3B as the sense strand. For reason ofcomparison an antisense oligonucleotide designated GB53 (Sternberger etal., supra) was generated which was directed against the PTEN mRNA aswell. The particularities of this latter experiment were as follows.

As may be taken from FIG. 3C end protected RNAi molecules depicted inFIG. 3B are functional in yielding a PTEN protein knockdown.

From this example it can be taken that both end protection groups renderRNAi molecules active in knocking down PTEN protein. This inhibition isas efficient as inhibition with antisense constructs but at lowerconcentrations used which is a clear advantage over the already verypowerful antisense technology.

Example 2: Overhang Requirements for RNAi Duplex Activity In Vivo

The experimental procedures were the same as depicted in connection withExample 1 except that the PTEN mRNA targeting interfering RNAi moleculeswere differently designed. The results are shown in FIG. 4A as doseresponse curves with FIG. 4B showing the particular sequence andmodifications of the interfering RNAi molecules used to generate thedata depicted in FIG. 4A. The nomenclature is such that, e.g., RNAi 18is composed of strand 18A as antisense strand and strand 18B as sensestrand.

Blunt ended molecules were compared to molecules with 3′-overhangs (RNAi18) and 5′-overhangs (RNAi 30 and RNAi 31) in their activity toknockdown PTEN mRNA in HeLa cells. The activity of blunt ended molecules(RNAi 28) and molecules with 5′-overhangs was comparable to the activityof molecules with 3′-overhangs. This shows that 3′-overhangs are notrequired for RNAi activity.

Example 3: Duplex Length Requirements of Interfering RNA Molecules forRNAi Activity In Vivo

The experimental approach was similar to the one outlined in connectionwith Example 1 except that the interfering RNA molecules were directedagainst the mRNA of Akt1. The negative control to show the specificityof the RNAi molecules was again p110 mRNA. The experimental results areshown in FIG. 5A with the particularities of the interfering RNAimolecules used being represented in FIG. 5B. Similar experiments werecarried out with further siRNA constructs which are depicted in FIG. 7A,left panel, whereby the arrows indicate mismatches anddeoxyribonucleotide are expressed in capital letters. The inhibition ofAkt1 mRNA expression in HeLa cells transfected with the indicatedamounts of siRNA molecules is depicted on the right panel of FIG. 7A.

Taqman analysis on Akt RNA from HeLa cells transfected with differentRNAi molecules shows that the double strand duplex of the siRNAmolecules has to be longer than 17 base pairs to show activity whereasmolecules with 17 base pair long duplexes or shorter are not functionaleven if sequence-specific overhangs are added. The shortest RNAimolecules successfully tested were 18 to 19 nucleotides or base pairs inlength. It is to be noted that the design of the interfering RNAmolecule 51A/51B referred to as RNAi 51 corresponds to the one asdescribed in international patent application WO 01/75164 The RNAimolecule 55A/55B comprises a stretch of 17 nucleotides and has a clearlydecreased activity in terms of degradation of Akt1 mRNA.

As may be taken from FIG. 7A 19 nt long duplexes are highly efficient inreducing Akt1 mRNA levels independent of the nature (deoxy- orribonucleotides) of the 3′ overhang (compare molecules 1AB, 2AB, 3AB,4AB). The 17 nucleotide long siRNA (molecule 5AB) showed a dramaticallyreduced silencing activity confirming the above expressed understandingthat active siRNA duplexes should be at least 18 nt or longer. Withoutwishing to be bound by any theory this result may be explainedmechanistically by two different requirements. First, a minimum basepairing of 18 nt between the antisense of the siRNA and the target mRNAmay be obligatory, or second, incorporation into the RNA-inducedsilencing complex (RISC) requires a minimum length of the siRNA duplex.To address this question a 19 nt long siRNA duplex molecule with one andtwo terminal mutations (CG and UA inversion) relative to the wild typesequence was synthesised (molecules 6AB and 7AB). Both molecules, eventhe molecule with a stretch of only 15 nt base pairing to the targetmRNA were functional in inducing the Akt1 mRNA level. Therefore, it canbe concluded that the duplex length itself, but not the base pairing ofthe antisense siRNA with the target mRNA seems to determine the minimumlength of functional siRNAs. This suggests that the length of thedouble-stranded helix is an important determinant for the incorporationinto the RISC complex. The introduced mismatches at the terminal ends ofthe siRNA duplexes had little effect on RNA interference.

Given the experimental results, the minimum requirement for optimum RNAimediated interference is thus a duplex length of 18 or 19 nucleotides,independent of the further design of the RNAi molecules such as bluntend or 5′-overhang or any other form as disclosed herein, but generallyapplicable to RNAi molecules. However, it has to be acknowledged thatthe particular design of the RNAi molecules may confer furtheradvantages to said molecules, such as, e.g., increased efficiency andincreased stability, respectively.

Example 4: Target-Antisense Homology Requirements for RNAi In Vivo

The experimental set-up was similar to the one described in example 1,whereby the RNAi is specific for Akt1. In addition, a PTEN specificinterfering RNA molecule was designed and used as negative control. Theresults are shown in FIG. 6A and FIG. 6B. Basically the same experimentwas carried out using further siRNA molecules as depicted in FIG. 7Bwith the results being indicated in FIG. 7B (right panel) and FIG. 7C,respectively.

Having established the minimal duplex length of 18 or more than 18nucleotides for functional siRNA molecules we asked the question howmany matching nucleotides between target mRNA and siRNA are necessaryfor silencing activity. As shown by Taqman analysis on Akt1 RNA astretch of 19 to 15 nucleotides perfectly matching to the target RNA, inthe case of Akt 1, was sufficient to mediate RNAi activity. A PTENspecific RNAi molecule did not reduce RNA amounts of Akt1 thusconfirming the specificity of this approach. Molecules containingmismatches of one or two nucleotides at any or both ends of a strandwere functional suggesting that a homolog stretch of 15 nt between atarget mRNA and RNAi is sufficient for gene silencing.

It can be concluded from these data that unspecific gene silencing canoccur by chance through unspecific binding to unrelated targets. This isbased on the understanding that a stretch of 15 to 17 matching basepairs is not specific for a single gene and will occur by chanceconsidering the complexity and size of the genome or transcriptosome ofvertebrates. Apart from the above mentioned experiments the location ofthe mismatch also was subsequently analysed. For this purpose a 19 ntlong blunt siRNA directed against PTEN mRNA was used. The sequencechanges in one siRNA strand were compensated by complementary changes inthe other strand to avoid disrupting duplex formation. As may be seenfrom both FIGS. 7B and C, respectively, a siRNA with only one pointmutation in the center of the molecule was severely compromised in itsability to use mRNA and protein expression levels. This result indicatesthat the RNA machinery is highly discriminative between perfect andimperfect base pairing between target mRNA and siRNA in the center ofthe duplex. This extreme dependence on a perfect complementarity betweentarget and siRNA has already been described for RNAi interference in theDrosophila system, however, not yet in connection with mammalian systemssuch as HeLa.

Based on this observation the present invention reduces this off-targetproblem of siRNA by two approaches: first, by reducing the moleculelength of the siRNA molecules to the minimal requirements (18-19 nt) andthereby reducing the chance of homology to off-targets, second, byinactivation of the sense strand to prevent an unwanted RNA silencingcaused by accidental complementarity of the sense strand to an unrelatedtarget RNA (see also Example 6).

Example 5: Stability of Modified RNAi Molecules in Serum

Oligonucleotides were incubated in human serum for 15 min and two hoursand loaded on 10% polyacrylamide gel with untreated controls. Theresults are shown in FIG. 8A. The various RNAi molecules used are shownand described in more detail in FIG. 8B.

From this example it can be taken that the RNAi duplex of RNA moleculeswith all nucleotides modified with 2′-O-methyl groups (RNAi molecules79A79B and 28A28B) have higher stability in serum. It is also shown thata blunt duplex is more stable than the duplex molecule with overhangs.From this the conclusion may be drawn that end protection (e.g. iB oramino) does not increase the stability in serum. In addition, it canalso be concluded that, in contrast to the understanding in the artbefore the filing of the present application, endonucleases are moreimportant than exonucleases in the protection of RNAi molecules.

In view of this, in addition to the various modifications or designs ofthe inventive RNAi molecules as disclosed above, further or additionalmodification of the nucleotides may include the use of aphosphorothioate backbone of the RNAi molecules which may be eithercomplete or partial in order to inhibit endonuclease function. Acomplete phosphorothioate backbone means that any of the nucleotidesexhibits a phosphorothioate group whereas a partial phosphorothioatebackbone means that not all of the nucleotides forming the RNAi moleculehave a phosphorothioate modification. This modification is suitable toincrease the lifetime of RNAi molecules irrespective of the furtherdesign of RNAi molecules. In this regard, the present inventionencompasses a partially or completely phosphorothioate modified RNAiwhich may be realized in connection with the different strategies forthe design of interfering RNA molecules as disclosed herein or with anyof the designs known in the art.

Example 6: Inactivation of the Sense Strand by NH₂ End Protection Groupson the 5′ and 3′ Ends

The experimental set-up was similar to the one described in connectionwith Example 1 with the target nucleic acid sequence being PTEN mRNA.The concentration of HeLa cells was 2,000 cells per well. RNA of PTENwas analysed in Taqman assays after transfection of differently modifiedRNAi molecules. The different interfering RNA molecules used aredepicted in FIG. 9B and the experimental results are shown in FIG. 9A.

The dose response curves of various RNAi molecules depicted in FIG. 8Ashow that RNAi molecules are functional when the sense strand, i.e. thesecond stand, is modified on both ends with amino groups. Particularlyeffective are RNAi molecules 20A26B, 18A26B, and 28A26B. The lowestactivity is shown by RNAi molecule 26A26B which corresponds to endmodification on all 4 ends of the duplex (the molecules described byTuschl are 18AB).

However, RNAi activity is also achieved when the antisense strand, i.e.the first strand, is modified only at the 3′ end leaving a free OH groupat the 5′ end (RNAi constructs 22A26B, 20A26B). There is no activitywhen the antisense strand is modified with amino groups on both the 5′and the 3′ end (26A26B). This leads to the conclusion that any end ofthe antisense strand and more particularly the 5′ end of the antisenseshould be kept without modifications. Additionally, it is worth statingthat the NH₂ end modification can be used to inactivate the sense strandon the 5′ and 3′ end and therefore reduce off-target effects mediated byan otherwise functional sense strand which results in a significantlyincreased specificity of the RNAi molecule which is advantageous fortarget validation as well as for any medical use of the RNAi molecule.

The further generalisation of the results from this experiment isdepicted in FIG. 9C. Functionally active RNAi are accordingly those nothaving an amino modification at the antisense strand or having an aminomodification only at the 3′ end of the antisense strand whereas an aminomodification at both ends of the antisense strand is not functional,i.e. does not result in a knockdown of the target mRNA.

Example 7: Impact of 2′-O-Methyl Modification of RNAi Molecules forEndonuclease Protection

RNA knockdown was again shown using real time RT-PCR analysis on HeLacells transfected with RNAi duplex molecules directed against the PTENmRNA as represented in FIG. 10A. Experimental procedures were basicallythe same as specified in Example 1. The structure of the RNAi moleculesinvestigated and their dose responses, which are depicted in FIG. 10A,are shown in FIG. 10C. The nucleotides printed in bold are those havinga 2′-O-methyl modification.

It is illustrated by the dose response curves shown for various RNAimolecules in FIG. 10A that internal 2′-O-alkyl groups reduce RNAiactivity. Preferably such 2′-O-alkyl groups are 2′-O-methyl or2′-O-ethyl groups. However, molecules with unmodified nucleotides incombination with 2′-O-alkyl modification show significant activity. Asis also depicted in FIG. 10A no activity was obtained when the antisensestrand is all modified with 2′-O-methyl groups and the sense strand isnot modified (c.f., e.g., RNAi molecule 79A28B). Taken the results of astability test such as incubation of the various RNAi molecules inserum, as depicted in FIG. 10B, shows that 2′-O-alkyl modificationsstabilize RNAi molecules against degradation. This clearly beneficialeffect, however, is at least to a certain degree counterbalanced by theeffect that 2′-O-alkyl modifications generally result in a reducedknockdown activity. Accordingly, the design of RNAi molecules has tobalance stability against activity which makes it important to be awareof the various design principles as disclosed in the presentapplication.

Example 8: Impact of Blocks of Internal 2′-O-Methyl Modifications on theStability of RNAi Molecules in Serum

The experimental approach in connection with this study was the same asdepicted in Example 1. Again, PTEN RNA was analysed by real time RT-PCRon HeLa cells at a density of 2000 cells/well which were transfectedwith different doses of RNAi molecules. RNAi molecules were incubated inserum for two hours and analysed on a 10% polyacrylamide gel. Theresults of this study are illustrated in FIGS. 11A to 11C, whereby FIG.11A shows the dose response of the various RNAi molecules depicted inFIG. 11C, and FIG. 11B shows the result of a stability test using someof the RNAi molecules depicted in FIG. 11C. The nucleotides written inbold in FIG. 11C are the ones carrying a modification, in this case a2′-O-methyl modification of the ribose moiety of the nucleotides.

A dose dependent inhibition by the unmodified RNAi molecules wasobserved. It was also shown that the 2′-O-methyl modification of thecore 9 nucleotides made the RNAi stable in serum and allowed activity ofthe duplex in mediating the interference phenomenon leading to adegradation of the PTEN mRNA. Total modification of the sense strandmakes the RNAi molecule stable in serum and allows certain activity.

Alternating blocks of 5 nucleotides with 2′-O-methyl modificationrenders the RNAi molecule stable in serum and allows activity on PTENRNA as shown by incubating the RNAi duplex in serum for two hours andloading the samples on a 10% polyacrylamide gel. As may be taken fromFIG. 11B the duplex comprising strands 80A and 80B was strongly degradedafter incubation in serum for two hours. The duplex consisting ofstrands 82A and 82B confirmed the result that the 5′-end of the firststrand which comprises the antisense strand should not be modified atthe 5′-terminal nucleotides (compare 82A82B with the reverse orientated81A81B). This was also confirmed by the results obtained having theduplex consisting of the strands 86A and 86B which is both active andstabilised in serum. It is noteworthy that molecules with unmodifiedblocks at the terminal 5′ of the antisense strand are more activewhereby the 5′terminal OH group is preferably not derivatized.

Further experiments were carried out using different modificationpatterns of 2′-O-methyl modification of the nucleotides. The resultsthereof are shown in FIG. 12A to 12C and further discussed herein inexample 9.

Example 9: The Impact of Alternating Internal 2′-O-Alkyl Modification onSerum Stability of RNAi Molecules

The experimental set-up for performing this kind of study was the sameas used in connection with the studies reported in Example 1 and Example8, respectively, with the targeted nucleic acid being again PTEN mRNA.HeLa cells were transfected with the different RNAi molecules depictedin FIG. 12B and RNA knockdown was demonstrated using real time RT-PCR onPTEN RNA in a dose-dependent manner (FIG. 12A). The stability of thevarious RNAi molecules after 15 min and two hours in serum at 37° C. isdepicted in FIG. 12C and a Western Blot for p110 and PTEN as thetarget-protein of the various RNAi molecules is depicted in FIG. 12Dwith the RNAi molecules tested being the same in both the experimentsunderlying FIG. 12C and FIG. 12D.

As illustrated in FIG. 12A and FIG. 12C nucleotides modified with2′-O-methyl groups alternating with unmodified nucleotides rendered RNAimolecules stable in serum while still allowing them to be active in thesense of interfering with the target mRNA. It was shown that incubationof RNAi duplex molecules for 15 min and two hours in serum degraded theunmodified duplex and the duplex where the 10 most 5′-positionednucleotides were unmodified.

In the RNAi molecules represented in FIG. 12B various patterns ofmodified and unmodified nucleotides were prepared. The RNAi molecule94A1/94B1 comprises a structure wherein a modified nucleotide is flankedby an unmodified nucleotide with the unmodified nucleotide being locatedat the 5′ end of the first strand. The RNAi molecule comprised ofstrands 94A2 and 94B2 was another example where the modified nucleotidesand the unmodified nucleotides of the first and the second strand werelocated at opposing sites. In contrast to this the RNAi moleculecomprised of strands 94A1 and 94B2 had the same pattern of modified andunmodified nucleotides. However, there was a phase shift such that themodified nucleotide base paired with an unmodified nucleotide. The twoRNAi molecules comprised of strands 94A1 and 94B1 and strands 94A2 and94B2 differed from each other such that in the first case the firststrand starts with an unmodified nucleotide and the corresponding firstnucleotide of the second strand, i.e. the nucleotide at the 3′ end ofthe second strand, starts with an unmodified nucleotide with thearrangement being opposite to this in the RNAi molecule comprised of94A2 and 94B2.

Additionally, alternatingly modified RNAi molecules as depicted in FIG.12B were functional in mediating a PTEN protein knock down as shown inFIG. 12D but only when the second 5′ and second 3′ terminal nucleotidewas not modified (see 94A294B1 and 94A294B2). Taken together these datashow that the most stable and most active RNAi molecules havealternating 2′ alkyl modified and unmodified nucleotide residues. Itshould be noted that these molecules do show a very similar mRNAreduction when compared to unmodified siRNA molecules while being stablein serum and therefore allowing increased or easier handling.

Example 10: Functional Protein Knockdown Mediated by Internally ModifiedRNAi Molecules

The experimental approach was similar to the one outlined in connectionwith example 1.

Western Blots were performed on HeLa cells harvested at various timepoints following transfection (48, 72, 96 and 120 hours) withalternatingly modified RNAi molecules as depicted in FIG. 12B. Forexperimental reasons it is noteworthy that at the 96 hour time pointcells were split and half the population was replated. A total of 40 nMof the various RNAi molecules were applied to the cells. Cells werecontinuously transfected for 72 h with cationic lipids as described inexample 1; then replated in the absence of transfection reagents.

Transfections were carried out in 96 well or 10-cm plates (at 30% to 50%confluency) by using various cationic lipids such as Oligofectamine,Lipofectamine (Life Technologies), NC388, L8 (Atugen, Berlin), RNAi weretransfected by adding pre-formed 5× concentrated complex of siRNAs andlipid in serum-free medium to cells in complete medium. The totaltransfection volume was 100 μl for cells plated in 96-wells and 10 mlfor cells in 10 cm plates. The final lipid concentration was 0.8 to 1.2μg/ml depending on cell density; the siRNA concentration is indicated ineach experiment.

The result of the Western Blot analysis is depicted in FIG. 13. As canbe taken from this Figure, modified RNAi molecules of the 94A2B1 and94A2B2 version yielded a longer lasting PTEN protein knock down asunmodified molecules. The lack of protein knock down also seen in FIG.12 with molecules of the 94A1B1 and 94A1B2 version was confirmed in thisexperiment. Unmodified molecules (80AB) were not as potent in supportinglong lasting protein knock down when the cells were not continuouslytransfected.

Example 11: Persistent PTEN Protein Knock Down with Alternating2′-O-Methyl Modifications of RNAi Molecules

The experimental approach was similar to the one outlined in connectionwith example 10 with the exception that the transfection was terminatedafter 5 h by replacing the transfection medium with new medium. Theprotocol was slightly modified such that for each of the RNAi moleculesa 40 nM concentration was realized using a stock solution of 1 μgRNAi/ml cationic lipid as described in connection with example 1. 5hours after transfection the medium was withdrawn and fresh EMEM added.The cells were split after 72 h, with half of the cells being lysed andthe other half newly plated and lysed 24 h later (96 h posttransfection). The result of a Western Blot analysis using 3 differentRNAi molecules (80AB, 94A1/B2, 94A2/B1 are depicted in FIG. 14. As apositive control untreated cells were used. FIG. 14 shows the expressionof PTEN after 72 h and 96 h, respectively. Taken the structuralparticularities of the various RNAi molecules it can be taken from FIG.14 that protein knockdown is persistent with alternating molecules ofthe 94A2B1 kind even over 96 h after splitting and replating cellscompared to unmodified RNAi molecules (such as 80AB) and RNAi molecule94A1B2.

A further experiment was carried out using the siRNA constructs asdepicted in FIG. 15A (left panel). From the results as depicted as ratioof PTEN/p110α mRNA degradation at the various concentrations of siRNAconstructs administered to the test system, it can be taken that siRNAmolecules with either one or both strands consisting of 2′-O-methylresidues were not able to induce RNA interference in the mammaliansystem (FIG. 15A, molecules V2, V5, V6). However, the decrease inactivity was less pronounced when only parts of the strands weremodified. Interestingly, a molecule having an unmodified antisensestrand (which is the upper strand in the representation throughout thisspecification if not indicated otherwise) and a completely modifiedsense strand was significantly more active when compared to the reversedversion (FIG. 5A, molecules V5 and V6). This result suggests that theantisense strand of the siRNA seems to be more critical and sensitive tomodification. The most efficient molecules inducing PTEN mRNA had onlystretches of modifications leaving the 5′ end unmodified or weremodified on alternating positions on both strands (FIG. 15A, moleculesV10, V12).

To test the nuclease resistance the different siRNA versions wereincubated in serum followed by PAA gel electrophoresis. The result isshown in FIG. 15B (right panel with the various sequences indicated onthe left panel of FIG. 15B). As shown earlier, blunt ended siRNAmolecules with unmodified ribonucleotides were very rapidly degradedwhereas a complete substitution with 2′-O-methyl nucleotides mediatedresistance against serum-derived nucleases (FIG. 15B, compare moleculeAB with V1). siRNA molecules with partial 2′-O-methyl modification alsoshowed an increased stability when compared to unmodified siRNAs. Inparticular, molecules with alternating modifications on both strandsshowed a significant improvement in instability (FIG. 15B, moleculesV13, V14, V15 and V12). More importantly, transfection of three of thesemolecules into HeLa cells resulted in a significant down regulation ofPTEN protein expression as depicted in FIG. 15C, length 6, 9 and 10). Inthis RNA interference activity assay an unexpected preference formolecules was observed which were modified at every second nucleotidebeginning with the most 5′ terminal nucleotide of the antisense strand(molecules V15 and V12). Molecules which contained modificationsbeginning with the second nucleotide at the 5′ end of the antisensestrand were more stable but had a strongly reduced activity in genesilencing (molecules V13, V14). This result points towards highlyspecific interactions between the involved enzymes and precisenucleotides in the siRNA duplex. Taken together the data shown hereindemonstrate that 2′-O-methyl modifications at particularly selectedpositions in the siRNA duplex can increase nuclease resistance and donot necessarily abolish RNAi completely.

Although an increased stability of synthetic siRNA has primarilyimplication for ill vivo application, it was also analysed whether theparticular modification can also lead to an extended protein knock-downin cell culture systems. Accordingly, HeLa cells were transientlytransfected for six hours using different versions of PTEN specificsiRNAs. The lipid siRNA complex was then washed away and the PTENprotein knock-down was analysed 48 hours and 120 hours later. Althoughknock-down experiments without continued transfection of siRNAs werecomplicated due to rapid growth of untransfected cells in this timeperiod resulting in a very transient knock-down, the present inventorswere able to demonstrate a prolonged PTEN protein knock-down with siRNAmolecules stabilised by the described 2′-O-methyl modification. At 48hours post transfection the unmodified siRNA (AB) showed the biggestreduction in PTEN protein levels, however, at 120 hours posttransfection the reduction in PTEN protein expression was superior withthe siRNA stabilised by alternating 2′-O-methyl modifications (FIG. 15D,compare lane 2 with lanes 4, 6 and 7).

From the results it can also be surmised that the starting nucleotideposition of the alternating modification can affect the activity of theduplex. To test this preference in more detail two additional series ofsiRNAs were synthesized, one specific for the kinase Akt1 and the otherone specific for p110β which is one of the two catalytic subunits ofPI(3-) kinase. The particular constructs are shown in FIG. 16A. Only 19nt long siRNA duplexes either without any or with 2′-O-methylmodification on every second other nucleotide were used. Using Akt1 as atarget an efficient protein knock-down as well as a dramatic reductionin phospho-Akt levels were observed with blunt, unmodified siRNAs (FIG.16A, right panel). From the different versions of molecules withmodifications on every second other nucleotide only one was efficientlymediating RNAi (FIG. 16A, molecule V5). This siRNA molecule contained anantisense strand which was modified at the most terminal 5′ and 3′nucleotides. The sense strand started with the unmodified nucleotides atthe most terminal position, resulting in a structure in which themodified and unmodified ribonucleotides of both strands face each other.As expected, this molecule was also protected against serum-derivednucleases as depicted in FIG. 16B (molecule V5).

Interestingly, a very similar 19 nt long siRNA molecule (V4) withmodifications beginning at the second nucleotide of the antisense strandshowed no RNA interference activation in the particular assay used. Theversion V6 in which the modified nucleotides of the antisense strandface modified nucleotides on the sense strand, was also inactive in thisexperiment. An identical series of 19 nt long siRNA molecules specificfor p110β confirmed these observations as depicted in FIG. 16C. Againthe similarly modified siRNA molecule (V5) was the most active, asindicated by reducing Akt phosphorylation, which is indicative for areduced P (I)-3 kinase activity due to reduced p110β levels. The reducedactivity of the molecules V6 might be explained by reduced duplexstability since the same structure was active in the PTEN knock-downexperiment with 21mer siRNAs. Although it is known that 2′-O-methylmodification on both strands facing each other reduces the stability ofnucleic acid duplexes, the difference between the activity of siRNAmolecules V4 and V5 (FIGS. 16B and C) probably is not due to differencesin duplex stability since the number of base pairing of modified andunmodified nucleotides is identical. This difference in activity mightbe due to specific requirements in the interacting proteins involved inthe degradation of the target mRNA. Also it can be taken from theseexperiments that the most terminal nucleotides of the antisense strandcan be modified at the 2′-OH-group with significant loss of silencingactivity.

Example 12: Different Loop Structures are Functional in Mediating RNAInterference

To test whether RNAi molecules, preferably synthetic RNAi molecules withself complementary structures, can inhibit gene expression asefficiently as standard double stranded siRNA molecules, HeLa cells weretransfected with p110β specific synthetic siRNAs. Transfections werecarried out in 96 well or 10-cm plates (at 30% to 50% confluency) byusing various cationic lipids such as Oligofectamine, Lipofectamine(Life Technologies), GeneBlocs were transfected by adding pre-formed 5×concentrated complex of GB and lipid in serum-free medium to cells incomplete medium. The total transfection volume was 100 μl for cellsplated in 96-wells and 10 ml for cells in 10 cm plates. The final lipidconcentration was 0.8 to 1.2 μg/ml depending on cell density; the RNAimolecule concentration is indicated in each experiment.

A dose dependent titration showed no significant difference inefficiency of mRNA knock-down achieved by the standard bimoleculardouble strand 21mer and the corresponding monomolecular molecules asanalysed by realtime PCR (Taqman) (FIG. 17A). Two different loopstructures a (A)₁₂ loop (SEQ ID NO: 175) and a HIV derived pA-loop weretested in parallel with similar results. A comparison of the relativeposition of the antisense sequence and the loop structure revealed animproved knock-down efficiency with the antisense sequence being located3′ to the loop (FIG. 17B; compare construct 3A, 3B and 4A, 4B).

Example 13: Studies on the Efficacy of Different Intramolecular HairpinLoops and Intermolecular “Bubbles”

The influence of different loop structures on inhibition of mRNA andprotein expression was tested. For these experiments Akt1 and Akt2 werechosen as targets. The experimental approach was similar to the onedescribed in example 12.

Significantly, the reduction in Akt1 mRNA as depicted in FIG. 18A andFIG. 18B as well as Akt1 protein levels as depicted in FIG. 18C wascompletely independent of the loop structure tested (compare molecules5A, 6A, 7A, 8A) (the structure of the RNAi molecule tested is alwaysdepicted underneath the bar diagram). Even a molecule containing arather unphysiological structure such as a polyethyleneglycol linker(PEG) as a loop efficiently reduced Akt1 expression indicating that sizeand nucleotide sequence of the loop is not crucial (FIG. 18A; molecule8A). A synthetic siRNA molecule specific for Akt2 (9A) was used tocontrol for specificity, and had no effect on Akt1 levels as shown inFIG. 15A. This molecule, however, efficiently silenced Akt2 expression(FIG. 18B; FIG. 18C). Self-complementary RNA molecules with loopstructures have the possibility to anneal as double strands inmonomolecular or bimolecular structures under physiologicalhybridization conditions (FIG. 18B, loop or bubble structure). Toaddress the question of whether the siRNA molecules exert their functionvia adapting an intra-molecular loop or an intermolecular “bubble”(schematically shown in FIG. 18B) two molecules not capable of foldingback on themselves were transfected. These constructs contained Akt1-and Akt2-specific sequences within the same molecule (FIG. 18B,constructs 10A, 10B) and were designed so as to prevent formation of anintramolecular duplex (i.e. a bimolecular duplex or “bubble” wasformed). Surprisingly, this molecule efficiently mediated both Akt1 andAkt2 mRNA knock-down as well as protein knock-down when transfectedafter annealing of both strands.

Whether loop and bubble structures are indeed substrates for RNAprocessing enzymes, e.g. Dicer, is not clear at this point. A recentstudy by Paddison and coworkers suggests that hairpin containing siRNAsare more dependent on Dicer activity than double stranded siRNAs.However, these data demonstrating RNA interference activity using a PEGlinker molecule indicate that the linker sequence is likely to beirrelevant.

The features of the present invention disclosed in the specification,the claims and/or the drawings may both separately and in anycombination thereof be material for realizing the invention in variousforms thereof.

The invention claimed is:
 1. A double-stranded siRNA molecule against atarget nucleic acid, wherein the double-stranded siRNA moleculecomprises a first strand and a second strand, wherein the first strandcomprises a first stretch that is complementary to the target nucleicacid, wherein the second strand comprises a second stretch that is thesame length as the first stretch and is complementary to the firststretch, wherein the first strand and the second strand form adouble-stranded structure comprising the first stretch and the secondstretch, wherein the first stretch and the second stretch each comprisescontiguous alternating 2′-O-alkyl ribonucleotides, wherein the2′-O-alkyl ribonucleotides alternate with unmodified or differentlymodified ribonucleotides, wherein the 2′-O-alkyl ribonucleotides of thefirst strand are shifted by one ribonucleotide relative to the2′-O-alkyl ribonucleotides of the second strand, and wherein each of thefirst stretch and the second stretch consists of at least 17 and fewerthan 30 ribonucleotides.
 2. The double-stranded siRNA molecule of claim1, wherein the double-stranded siRNA molecule is blunt ended on bothends.
 3. The double-stranded siRNA molecule of claim 1, wherein at leastone end of the double-stranded siRNA molecule has an overhang.
 4. Thedouble-stranded siRNA molecule of claim 3, wherein the overhang has oneto eight nucleotides.
 5. The double-stranded siRNA molecule of claim 3,wherein the 3′ end of the first strand has an overhang.
 6. Thedouble-stranded siRNA molecule of claim 3, wherein the 5′ end of thefirst strand has an overhang.
 7. The double-stranded siRNA molecule ofclaim 3, wherein the 3′ end of the second strand has an overhang.
 8. Thedouble-stranded siRNA molecule of claim 3, wherein the 5′ end of thesecond strand has an overhang.
 9. The double-stranded siRNA molecule ofclaim 1, wherein each of the first strand and the second strand consistsof at least 18 nucleotides.
 10. The double-stranded siRNA molecule ofclaim 1, wherein the 2′-O-alkyl ribonucleotides are 2′-O-methylribonucleotides.
 11. The double-stranded siRNA molecule of claim 10,wherein the 2′-O-methyl ribonucleotides alternate with the unmodifiedribonucleotides.
 12. The double-stranded siRNA molecule of claim 1,wherein the differently modified ribonucleotides have an amino, fluoro,different alkoxy, or alkyl modification at the 2′-position.
 13. Thedouble-stranded siRNA molecule of claim 10, wherein the 2′-O-methylribonucleotides alternate with 2′-fluoro ribonucleotides.
 14. Thedouble-stranded siRNA molecule of claim 1, wherein the first stretch andthe second stretch each comprises contiguous alternating single2′-O-alkyl ribonucleotides, and wherein the single 2′-O-alkylribonucleotides alternate with the unmodified ribonucleotides.
 15. Thedouble-stranded siRNA molecule of claim 14, wherein the single2′-O-alkyl ribonucleotides are 2′-O-methyl ribonucleotides.
 16. Thedouble-stranded siRNA molecule of claim 1, wherein the first stretch andthe second stretch each comprises contiguous alternating single2′-O-alkyl ribonucleotides, and wherein the single 2′-O-alkylribonucleotides alternate with the differently modified ribonucleotides.17. The double-stranded siRNA molecule of claim 16, wherein the single2′-O-alkyl ribonucleotides are 2′-O-methyl ribonucleotides.
 18. Thedouble-stranded siRNA molecule of claim 16, wherein the differentlymodified ribonucleotides have an amino, fluoro, different alkoxy, oralkyl modification at the 2′-position.
 19. The double-stranded siRNAmolecule of claim 16, wherein the single 2′-O-alkyl ribonucleotides are2′-O-methyl ribonucleotides, and the differently modifiedribonucleotides are 2′-fluoro ribonucleotides.
 20. The double-strandedsiRNA molecule of claim 1, wherein the 5′ ribonucleotide of the firststretch is a 2′-O-alkyl modified ribonucleotide.
 21. The double-strandedsiRNA molecule of claim 11, wherein the 5′ ribonucleotide of the firststretch is a 2′-O-methyl ribonucleotide.
 22. The double-stranded siRNAmolecule of claim 13, wherein the 5′ ribonucleotide of the first stretchis a 2′-O-methyl ribonucleotide.
 23. The double-stranded siRNA moleculeof claim 14, wherein the 5′ ribonucleotide of the first stretch is a2′-O-alkyl modified ribonucleotide that alternates with an unmodifiedribonucleotide.
 24. The double-stranded siRNA molecule of claim 16,wherein the 5′ ribonucleotide of the first stretch is a 2′-O-alkylmodified ribonucleotide that alternates with a differently modifiedribonucleotide.
 25. The double-stranded siRNA molecule of claim 19,wherein the double-stranded siRNA molecule is blunt on at least one end.26. The double-stranded siRNA molecule of claim 19, wherein at least oneend of the double-stranded siRNA molecule has an overhang of one toeight nucleotides.
 27. The double-stranded siRNA molecule of claim 26,wherein the 3′ end of the first strand has an overhang.
 28. Thedouble-stranded siRNA molecule of claim 26, wherein the 5′ end of thefirst strand has an overhang.
 29. The double-stranded siRNA molecule ofclaim 26, wherein the 3′ end of the second strand has an overhang. 30.The double-stranded siRNA molecule of claim 26, wherein the 5′ end ofthe second strand has an overhang.